Use of dimerization domain component stacks to modulate plant architecture

ABSTRACT

This invention provides means for altering the harvest index of crop plants by modulating the expression of transgenic genes using dimerization domain and component stacks, thereby modulating plant architecture. The transgene/dimerization domain stacks are provided in a single transformation vector unit and are used to modulate plant growth, yield, and harvest index in plants.

CROSS REFERENCE

This utility application is a continuation of, and claims the benefit ofco-pending U.S. non provisional application Ser. No. 12/837,553, filed16 Jul. 2010, and further claims the benefit U.S. ProvisionalApplication Ser. Nos. 61/228,195 and 61/286,061, filed Jul. 24, 2009 andDec. 14, 2009 respectively, which are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates generally to the field of molecular biology.

BACKGROUND OF THE INVENTION

Harvest index, ratio of grain to total above ground biomass, hasremained nearly constant around 50% in maize over the past 100 years(Sinclair, (1998) Crop Science 38:638-643; Tollenaar and Wu, (1999)“Crop Science 39:1597-1604). Thus, the quadrupling of grain yield overthe last 50-60 years has resulted from an increase in total biomassproduction per unit land area, which has been accomplished by increasedplanting density (Duvick and Cassman, (1999) Crop Science 39:1622-1630).Selection for higher grain yield under increasing planting densities hasled to a significant architectural change in plant structure that ofrelatively erect and narrow leaves to minimize shading. An undesirableconsequence of higher density planting (or higher plant populations) hasbeen the increased frequency of stalk and root lodging. The relationshipbetween planting density and biomass production deviates significantlyfrom linearity as the optimal density is approached for maximal biomassyield per unit land area. This is reflected in a proportionately greaterreduction in the individual plant biomass, which manifests in the formof weaker stalks and hence increased lodging. In addition, approximately20% of total biomass at maturity stays in the form of roots in the soil,contributing to its organic matter content (Amos and Walters, 2006).Since both stalk and root lodging are agronomic characteristicsaffecting harvest index, dwarf type plants could have potentialadvantages in yield stability.

Dwarf plants have had a major impact on agriculture. Dwarf varieties ofwheat (and other small grain cereals) are widely used in North Americadue to both reduced potential for lodging and response to more intensivemanagement and yield stability and potentially higher yields. There areother benefits that may be realized from the higher harvest index ofdwarf crop plants including reductions in the amounts of pesticides andfertilizers required, higher planting densities and reduced labor costs.Dwarf plants provide ease in harvesting, simplified management of cropsand potential reductions in water and nutrient use.

In view of the current trends of both increasing human population andthe decreasing land area suitable for agriculture, increasingagricultural productivity is, and will continue to be, a challenge ofparamount importance. Dwarf crop plants are important components of ouragricultural production system. Increased usage of dwarf crop plants mayhelp to meet the agricultural production demands of the future.

Genes that increase stalk strength, i.e., Cellulose Synthase, areresponsible for cellulose production in crop plants, can be modified toincrease size and strength of various plant tissues. Cellulose in a unitlength of the maize stalk was found to be the best indicator ofmechanical strength (Appenzeller, et al., (2004) Cellulose 11:287-299;Ching, et al., (2006)). Increasing cellulose concentration in the stalkdry matter could lead to improving stalk mechanical strength andincreasing biomass which in turn increases yield and potentially harvestindex. Improvements in plant strength (biomass) and growth of specificplant tissues (organs) provides plants with greater biomass andincreased harvest index.

Flowering time determines maturity, an important agronomic trait. Genesthat control the transition from vegetative to reproductive growth areessential for manipulation of flowering time. In maize, flowering genesprovide opportunities for enhanced crop yield, adaptation of germplasmto different climatic zones and synchronous flowering for hybrid seedproduction. The development of inbred lines having modified floweringfacilitates the movement of elite germplasm across maturity zones. Inaddition, additional opportunities exist to increase the rate of grainfill and/or grain dry down to complement changes in the onset offlowering.

The combined controlled expression of plant architecture genes,flowering time genes and dwarfing gene components within transformedplants would not only increase the yield potential and harvest index ofcrop plants but would also improve the agronomic characteristics thatsimplify management practices and increase the adaptation of cropspecies into new geographic areas.

This invention provides means for altering the harvest index of cropplants by modulating the expression of transgenes using multiple stackedplant genes and dwarf gene components, thereby modulating plantarchitecture. A component of Dwarf gene D8, the dimerization domain(DD), a leucine-zipper dimerization domain (SEQ ID NO: 9) isoverexpressed as a dominant negative transgene. Thetransgene/dimerization domain component stacks are provided in a singletransformation vector unit and are used to modulate specific plantorgans of a plant that can increase growth, yield and harvest index inplants. The expression in specific plant tissues, such as roots, ears ortassels can lead to elongation of the specific plant organs.

These stacked units could be used to enhance crop plant performance andvalue in several areas including: 1) plant standability (composed ofstalk and root lodging), harvest index and yield potential; 2)modification of specific plant organ size; 3) plant dry matter as afeedstock for ethanol or for other renewable bioproducts and 4) silage.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods for controlling plant growth and dimerizationdomain component stack formation for increasing yield in a plant areprovided. The compositions include dimerization domain component stacksfrom maize. Compositions of the invention comprise amino acid sequencesand nucleotide sequences selected from SEQ ID NOS: 1-22 as well asvariants and fragments thereof.

Polynucleotides encoding the dimerization domain component stacks areprovided in DNA constructs for expression in a plant of interest.Expression cassettes, plants, plant cells, plant parts and seedscomprising the sequences of the invention are further provided. Inspecific embodiments, the polynucleotide is operably linked to aconstitutive promoter.

Methods for modulating the level of a dimerization domain componentstack sequence in a plant or plant part are provided. The methodscomprise introducing into a plant or plant part a heterologouspolynucleotide comprising a dimerization domain component stack sequenceof the invention. The level of a dimerization domain component stackpolypeptide can be increased or decreased. Such method can be used toincrease the yield in plants; in one embodiment, the method is used toincrease grain yield in cereals.

The plant hormone GA is active in various growth processes, specificallythe elongation of stem and root during plant growth. The D8 (and D9)genes of maize encode for transcriptional regulators that act asinhibitors of the giberellic acid signal transduction pathway, andconsist of a DELLA and GRAS domain. The GA receptor interacts with DELLAproteins in the presence of GA, which leads to poly-ubiquitination ofthe DELLA protein. Poly-ubiquination signals for protein degradation bythe 26S proteasome. The degradation of the DELLA proteins removes theirinhibition of the GA growth response. In general, the rate ofdegradation of the D8/D9 proteins appears to correlate with plant size(i.e. slower degradation results in less response to GA, less elongationand a greater height reduction). Deletions and specific mutations in theDELLA domain of D8 are responsible for the dwarfing phenotype because ofthe altered degradation kinetics of these proteins.

The D8 (and D9) proteins are thought to function in-vivo as a dimer,whose catabolism regulates plant elongation. Dimers of strong dwarfgenes such as D8 are less sensitive to degradation while moderate dwarfgenes such as D8MPL are relatively more sensitive to degradation. Thenative wild type gene d8 is sensitive to degradation and a tall ornormal height is observed. A specific leucine-zipper domain of the D8protein, ZM-D8 243-331, is involved in the formation of the dimers. Analtered dimerization domain protein is formed by over expression of theZM-D8 243-331 protein. These truncated protein fragments compete forbinding to the leucine-zipper domain of full length D8 and D9. Thiscompetitive binding leads to the formation of defective dimers having afull-length protein::truncated protein. The resultant non-functionaldimer lacks the capacity to inhibit the GA response, and when present ina plant or plant organ increases elongation. Further, tissue specificexpression using promoters for specific plant organs such as roots, earsor tassels are expected to have increased size (length) compared todwarf plants. Specifically, a dwarf plant type could have roots that aresimilar in size to wild type or normal statured plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Root Growth as measured in Mini Rhizitrons in Johnston Iowa in2006 with Hybrid 33A14, PHP24843, PHP26998 and PHP26998

FIG. 2: D8-MPL Stack Average Harvest Index by construct, based on lateseason plant dry weight.

FIG. 3: D8-MPL Stack Yield Comparison at 24K

FIG. 4: Diagram describing selective architecture modification of Zm-D8243-331, a dominant negative transgene, overexpression of DD, leading tonon-functional dimers. Non-functional Dimers (DN) increase elongationwhen expressed in tissues such as roots, ears or tassels.

FIG. 5: Alignment of DD domains across various species, Glycine max (SEQID NOS: 24, 26, 28 30, Arabidopsis thaliana (SEQ ID NOS: 32, 34, 36, 38and 40), Zea mays (SEQ ID NO: 19), showing conserved regions andconsensus sequence (SEQ ID NO: 41).

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Unless mentioned otherwise, thetechniques employed or contemplated herein are standard methodologieswell known to one of ordinary skill in the art. The materials, methodsand examples are illustrative only and not limiting. The following ispresented by way of illustration and is not intended to limit the scopeof the invention.

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the invention are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of botany, microbiology, tissueculture, molecular biology, chemistry, biochemistry and recombinant DNAtechnology, which are within the skill of the art. Such techniques areexplained fully in the literature. See, e.g., Langenheim and Thimann,BOTANY: PLANT BIOLOGY AND ITS RELATION TO HUMAN AFFAIRS, John Wiley(1982); CELL CULTURE AND SOMATIC CELL GENETICS OF PLANTS, vol. 1, Vasil,ed. (1984); Stanier, et al., THE MICROBIAL WORLD, 5^(th) ed.,Prentice-Hall (1986); Dhringra and Sinclair, BASIC PLANT PATHOLOGYMETHODS, CRC Press (1985); Maniatis, et al., MOLECULAR CLONING: ALABORATORY MANUAL (1982); DNA CLONING, vols. I and II, Glover, ed.(1985); OLIGONUCLEOTIDE SYNTHESIS, Gait, ed. (1984); NUCLEIC ACIDHYBRIDIZATION, Hames and Higgins, eds. (1984) and the series METHODS INENZYMOLOGY, Colowick and Kaplan, eds, Academic Press, Inc., San Diego,Calif.

Units, prefixes and symbols may be denoted in their SI accepted form.Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively. Numeric ranges are inclusiveof the numbers defining the range. Amino acids may be referred to hereinby either their commonly known three letter symbols or by the one-lettersymbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The terms defined below are more fullydefined by reference to the specification as a whole.

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

By “microbe” is meant any microorganism (including both eukaryotic andprokaryotic microorganisms), such as fungi, yeast, bacteria,actinomycetes, algae and protozoa, as well as other unicellularstructures.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), 0-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., DIAGNOSTIC MOLECULARMICROBIOLOGY: PRINCIPLES AND APPLICATIONS, Persing, et al., eds.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

The term “conservatively modified variants” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refer to those nucleic acidsthat encode identical or conservatively modified variants of the aminoacid sequences. Because of the degeneracy of the genetic code, a largenumber of functionally identical nucleic acids encode any given protein.For instance, the codons GCA, GCC, GCG and GCU all encode the amino acidalanine. Thus, at every position where an alanine is specified by acodon, the codon can be altered to any of the corresponding codonsdescribed without altering the encoded polypeptide. Such nucleic acidvariations are “silent variations” and represent one species ofconservatively modified variation. Every nucleic acid sequence hereinthat encodes a polypeptide also describes every possible silentvariation of the nucleic acid. One of ordinary skill will recognize thateach codon in a nucleic acid (except AUG, which is ordinarily the onlycodon for methionine; one exception is Micrococcus rubens, for which GTGis the methionine codon (Ishizuka, et al., (1993) J. Gen. Microbiol.139:425-32) can be modified to yield a functionally identical molecule.Accordingly, each silent variation of a nucleic acid, which encodes apolypeptide of the present invention, is implicit in each describedpolypeptide sequence and incorporated herein by reference.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” when the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Thus, any number of amino acid residues selected from the group ofintegers consisting of from 1 to 15 can be so altered. Thus, forexample, 1, 2, 3, 4, 5, 7 or 10 alterations can be made. Conservativelymodified variants typically provide similar biological activity as theunmodified polypeptide sequence from which they are derived. Forexample, substrate specificity, enzyme activity, or ligand/receptorbinding is generally at least 30%, 40%, 50%, 60%, 70%, 80% or 90%,preferably 60-90% of the native protein for it's native substrate.Conservative substitution tables providing functionally similar aminoacids are well known in the art.

The following six groups each contain amino acids that are conservativesubstitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V) and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See also, Creighton, PROTEINS, W.H. Freeman and Co. (1984).

As used herein in the context of nucleic acids in general, “consistingessentially of” means the inclusion of additional sequences to an objectpolynucleotide where the additional sequences do not selectivelyhybridize, under stringent hybridization conditions, to the same cDNA asthe polynucleotide and where the hybridization conditions include a washstep in 0.1×SSC and 0.1% sodium dodecyl sulfate at 65° C.

The term “consisting essentially of” or “consists essentially of” in thecontext of a nucleic acid sequence encoding a dimerization domain or theamino acid sequence of the dimerization domain, generally refers to arecombinant dimerization domain sequence and any other sequence thatdoes not materially alter the basic binding property of the dimerizationdomain fragment, for example, to form a defective dimer with the targetprotein. For example, the ZM-D8 243-331 is a portion of the D8 proteinthat corresponds to a dimerization domain region. In an embodiment, thisdomain fragment may contain other sequences both to the amino and/orcarboxy-terminus as long as the additional sequences do not materiallyalter the basic binding characteristics of the dimerization domainfragment with the target protein that results in reduced inhibition bygiberrellic acid (GA) hormone. For example, a full-length D8 amino acidsequence is not suitable as it will result in the formation of afunctional dimer that blocks GA response.

By “encoding” or “encoded,” with respect to a specified nucleic acid, ismeant comprising the information for translation into the specifiedprotein. A nucleic acid encoding a protein may comprise non-translatedsequences (e.g., introns) within translated regions of the nucleic acid,or may lack such intervening non-translated sequences (e.g., as incDNA). The information by which a protein is encoded is specified by theuse of codons. Typically, the amino acid sequence is encoded by thenucleic acid using the “universal” genetic code. However, variants ofthe universal code, such as is present in some plant, animal and fungalmitochondria, the bacterium Mycoplasma capricolum (Yamao, et al., (1985)Proc. Natl. Acad. Sci. USA 82:2306-2309) or the ciliate Macronucleus,may be used when the nucleic acid is expressed using these organisms.

When the nucleic acid is prepared or altered synthetically, advantagecan be taken of known codon preferences of the intended host where thenucleic acid is to be expressed. For example, although nucleic acidsequences of the present invention may be expressed in bothmonocotyledonous and dicotyledonous plant species, sequences can bemodified to account for the specific codon preferences and GC contentpreferences of monocotyledonous plants or dicotyledonous plants as thesepreferences have been shown to differ (Murray, et al., (1989) NucleicAcids Res. 17:477-98 and herein incorporated by reference). Thus, themaize preferred codon for a particular amino acid might be derived fromknown gene sequences from maize. Maize codon usage for 28 genes frommaize plants is listed in Table 4 of Murray, et al., supra.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

By “host cell” is meant a cell, which contains a vector and supports thereplication and/or expression of the expression vector. Host cells maybe prokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, plant, amphibian or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells, including but notlimited to maize, sorghum, sunflower, soybean, wheat, alfalfa, rice,cotton, canola, barley, millet and tomato. A particularly preferredmonocotyledonous host cell is a maize host cell.

The term “hybridization complex” includes reference to a duplex nucleicacid structure formed by two single-stranded nucleic acid sequencesselectively hybridized with each other.

The term “introduced” in the context of inserting a nucleic acid into acell, means “transfection” or “transformation” or “transduction” andincludes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

The terms “isolated” refers to material, such as a nucleic acid or aprotein, which is substantially or essentially free from componentswhich normally accompany or interact with it as found in its naturallyoccurring environment. The isolated material optionally comprisesmaterial not found with the material in its natural environment. Nucleicacids, which are “isolated”, as defined herein, are also referred to as“heterologous” nucleic acids. Unless otherwise stated, the term“dimerization domain component stack nucleic acid” means a nucleic acidcomprising a polynucleotide (“dimerization domain component stackpolynucleotide”) encoding a dimerization domain component stackpolypeptide.

As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

By “nucleic acid library” is meant a collection of isolated DNA or RNAmolecules, which comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism. Constructionof exemplary nucleic acid libraries, such as genomic and cDNA libraries,is taught in standard molecular biology references such as Berger andKimmel, GUIDE TO MOLECULAR CLONING TECHNIQUES, from the series METHODSIN ENZYMOLOGY, vol. 152, Academic Press, Inc., San Diego, Calif. (1987);Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2^(nd) ed.,vols. 1-3 (1989) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, etal., eds, Current Protocols, a joint venture between Greene PublishingAssociates, Inc. and John Wiley & Sons, Inc. (1994 Supplement).

As used herein “operably linked” includes reference to a functionallinkage between a first sequence, such as a promoter and a secondsequence, wherein the promoter sequence initiates and mediatestranscription of the DNA sequence corresponding to the second sequence.Generally, operably linked means that the nucleic acid sequences beinglinked are contiguous and, where necessary to join two protein codingregions, contiguous and in the same reading frame.

As used herein, the term “plant” includes reference to whole plants,plant organs (e.g., leaves, stems, roots, etc.), seeds and plant cellsand progeny of same. Plant cell, as used herein includes, withoutlimitation, seeds suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollenand microspores. The class of plants, which can be used in the methodsof the invention, is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants including species from the genera: Cucurbita,Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium,Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus,Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis,Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Avena, Hordeum, Secale, Allium and Triticum.Also included are grass plants from the Poaceae family including but notlimited to the genera: Poa, Agrostis, Lolium, Festuca, Zoysia, Cynodon,Stenotaphrum, Paspalum, Eremochloa, Axonopus, Buchloe, Bouteloua,including Bluegrass, Bentgrass, Ryegrasses, Fescues, Zoysiagrass,Bermudagrass, St. Augustine grass, Bahiagrass, Centipedegrass,Carpetgrass, Buffalograss and Gramagrass. A particularly preferred plantis Zea mays.

As used herein, “yield” includes reference to bushels per acre of agrain crop at harvest, as adjusted for grain moisture (15% typically).Grain moisture is measured in the grain at harvest. The adjusted testweight of grain is determined to be the weight in pounds per bushel,adjusted for grain moisture level at harvest.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide or analogs thereof that havethe essential nature of a natural ribonucleotide in that they hybridize,under stringent hybridization conditions, to substantially the samenucleotide sequence as naturally occurring nucleotides and/or allowtranslation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including inter alia, simple andcomplex cells.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers.

As used herein “promoter” includes reference to a region of DNA upstreamfrom the start of transcription and involved in recognition and bindingof RNA polymerase and other proteins to initiate transcription. A “plantpromoter” is a promoter capable of initiating transcription in plantcells. Exemplary plant promoters include, but are not limited to, thosethat are obtained from plants, plant viruses and bacteria which comprisegenes expressed in plant cells such Agrobacterium or Rhizobium. Examplesare promoters that preferentially initiate transcription in certaintissues, such as leaves, roots, seeds, fibres, xylem vessels, tracheidsor sclerenchyma. Such promoters are referred to as “tissue preferred.” A“cell type” specific promoter primarily drives expression in certaincell types in one or more organs, for example, vascular cells in rootsor leaves. An “inducible” or “regulatable” promoter is a promoter, whichis under environmental control. Examples of environmental conditionsthat may effect transcription by inducible promoters include anaerobicconditions or the presence of light. Another type of promoter is adevelopmentally regulated promoter, for example, a promoter that drivesexpression during pollen development. Tissue preferred, cell typespecific, developmentally regulated and inducible promoters constitutethe class of “non-constitutive” promoters. A “constitutive” promoter isa promoter, which is active under most environmental conditions.

The term “dimerization domain component stack polypeptide” refers to oneor more amino acid sequences that include the dimerization domain regionof interest and another polypeptide sequence that is not the same parentsequence from which the dimerization domain sequence was derived. Theterm is also inclusive of fragments, variants, homologs, alleles orprecursors (e.g., preproproteins or proproteins) thereof. A“dimerization domain component stack protein” comprises a dimerizationdomain component stack polypeptide. Unless otherwise stated, the term“dimerization domain component stack nucleic acid” means a nucleic acidcomprising a polynucleotide (“dimerization domain component stackpolynucleotide”) encoding a dimerization domain component stackpolypeptide.

As used herein “recombinant” includes reference to a cell or vector,that has been modified by the introduction of a heterologous nucleicacid or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found in identicalform within the native (non-recombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, under expressed ornot expressed at all as a result of deliberate human intervention. Theterm “recombinant” as used herein does not encompass the alteration ofthe cell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without deliberate human intervention. The term“recombinant polypeptide” or “recombinant nucleic acid” refers to thepeptide and nucleic acid sequences that have been modified such thatthey do not exist in nature in their present form.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements, which permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed and apromoter.

The term “residue” or “amino acid residue” or “amino acid” are usedinterchangeably herein to refer to an amino acid that is incorporatedinto a protein, polypeptide or peptide (collectively “protein”). Theamino acid may be a naturally occurring amino acid and, unless otherwiselimited, may encompass known analogs of natural amino acids that canfunction in a similar manner as naturally occurring amino acids.

The term “selectively hybridizes” includes reference to hybridization,under stringent hybridization conditions, of a nucleic acid sequence toa specified nucleic acid target sequence to a detectably greater degree(e.g., at least 2-fold over background) than its hybridization tonon-target nucleic acid sequences and to the substantial exclusion ofnon-target nucleic acids. Selectively hybridizing sequences typicallyhave about at least 40% sequence identity, preferably 60-90% sequenceidentity and most preferably 100% sequence identity (i.e.,complementary) with each other.

The terms “stringent conditions” or “stringent hybridization conditions”include reference to conditions under which a probe will hybridize toits target sequence, to a detectably greater degree than other sequences(e.g., at least 2-fold over background).

Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences can beidentified which can be up to 100% complementary to the probe(homologous probing). Alternatively, stringency conditions can beadjusted to allow some mismatching in sequences so that lower degrees ofsimilarity are detected (heterologous probing). Optimally, the probe isapproximately 500 nucleotides in length, but can vary greatly in lengthfrom less than 500 nucleotides to equal to the entire length of thetarget sequence.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide or Denhardt's.Exemplary low stringency conditions include hybridization with a buffersolution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1%SDS at 37° C. and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary highstringency conditions include hybridization in 50% formamide, 1 M NaCl,1% SDS at 37° C. and a wash in 0.1×SSC at 60 to 65° C. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA-DNA hybrids, the T_(m) can be approximated from theequation of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-84:T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with ≧90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3 or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9 or 10° C. lower than the thermalmelting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15 or 20° C. lower than thethermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY—HYBRIDIZATION WITH NUCLEIC ACID PROBES, part I, chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier, New York (1993) and CURRENT PROTOCOLS INMOLECULAR BIOLOGY, chapter 2, Ausubel, et al., eds, Greene Publishingand Wiley-Interscience, New York (1995). Unless otherwise stated, in thepresent application high stringency is defined as hybridization in4×SSC, 5×Denhardt's (5 g Ficoll, 5 g polyvinypyrrolidone, 5 g bovineserum albumin in 500 ml of water), 0.1 mg/ml boiled salmon sperm DNA,and 25 mM Na phosphate at 65° C. and a wash in 0.1×SSC, 0.1% SDS at 65°C.

As used herein, “transgenic plant” includes reference to a plant, whichcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,non-recombinant viral infection, non-recombinant bacterialtransformation, non-recombinant transposition or spontaneous mutation.

As used herein, “vector” includes reference to a nucleic acid used intransfection of a host cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides or polypeptides:(a) “reference sequence,” (b) “comparison window,” (c) “sequenceidentity,” (d) “percentage of sequence identity” and (e) “substantialidentity.”

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence or the complete cDNA or gene sequence.

As used herein, “comparison window” means includes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence may be compared to a reference sequence andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. Generally, the comparison windowis at least 20 contiguous nucleotides in length, and optionally can be30, 40, 50, 100 or longer. Those of skill in the art understand that toavoid a high similarity to a reference sequence due to inclusion of gapsin the polynucleotide sequence a gap penalty is typically introduced andis subtracted from the number of matches.

Methods of alignment of nucleotide and amino acid sequences forcomparison are well known in the art. The local homology algorithm(BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math 2:482, mayconduct optimal alignment of sequences for comparison; by the homologyalignment algorithm (GAP) of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-53; by the search for similarity method (Tfasta and Fasta) ofPearson and Lipman, (1988) Proc. Natl. Acad. Sci. USA 85:2444; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package®, Version 8 (available from Genetics ComputerGroup (GCG® programs (Accelrys, Inc., San Diego, Calif.)). The CLUSTALprogram is well described by Higgins and Sharp, (1988) Gene 73:237-44;Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et al., (1988) NucleicAcids Res. 16:10881-90; Huang, et al., (1992) Computer Applications inthe Biosciences 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol.24:307-31. The preferred program to use for optimal global alignment ofmultiple sequences is PileUp (Feng and Doolittle, (1987) J. Mol. Evol.,25:351-60 which is similar to the method described by Higgins and Sharp,(1989) CABIOS 5:151-53 and hereby incorporated by reference). The BLASTfamily of programs which can be used for database similarity searchesincludes: BLASTN for nucleotide query sequences against nucleotidedatabase sequences; BLASTX for nucleotide query sequences againstprotein database sequences; BLASTP for protein query sequences againstprotein database sequences; TBLASTN for protein query sequences againstnucleotide database sequences and TBLASTX for nucleotide query sequencesagainst nucleotide database sequences. See, CURRENT PROTOCOLS INMOLECULAR BIOLOGY, Chapter 19, Ausubel, et al., eds., Greene Publishingand Wiley-Interscience, New York (1995).

GAP uses the algorithm of Needleman and Wunsch, supra, to find thealignment of two complete sequences that maximizes the number of matchesand minimizes the number of gaps. GAP considers all possible alignmentsand gap positions and creates the alignment with the largest number ofmatched bases and the fewest gaps. It allows for the provision of a gapcreation penalty and a gap extension penalty in units of matched bases.GAP must make a profit of gap creation penalty number of matches foreach gap it inserts. If a gap extension penalty greater than zero ischosen, GAP must, in addition, make a profit for each gap inserted ofthe length of the gap times the gap extension penalty. Default gapcreation penalty values and gap extension penalty values in Version 10of the Wisconsin Genetics Software Package® are 8 and 2, respectively.The gap creation and gap extension penalties can be expressed as aninteger selected from the group of integers consisting of from 0 to 100.Thus, for example, the gap creation and gap extension penalties can be0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity and Similarity. The Quality is the metric maximized in order toalign the sequences. Ratio is the quality divided by the number of basesin the shorter segment. Percent Identity is the percent of the symbolsthat actually match. Percent Similarity is the percent of the symbolsthat are similar. Symbols that are across from gaps are ignored. Asimilarity is scored when the scoring matrix value for a pair of symbolsis greater than or equal to 0.50, the similarity threshold. The scoringmatrix used in Version 10 of the Wisconsin Genetics Software Package® isBLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA89:10915).

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the BLAST 2.0 suite of programsusing default parameters (Altschul, et al., (1997) Nucleic Acids Res.25:3389-402).

As those of ordinary skill in the art will understand, BLAST searchesassume that proteins can be modeled as random sequences. However, manyreal proteins comprise regions of nonrandom sequences, which may behomopolymeric tracts, short-period repeats or regions enriched in one ormore amino acids. Such low-complexity regions may be aligned betweenunrelated proteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs can be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Claverie andStates, (1993) Comput. Chem. 17:191-201) low-complexity filters can beemployed alone or in combination.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences includes reference to the residuesin the two sequences, which are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. Where sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences, which differ by suchconservative substitutions, are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., according to the algorithm of Meyersand Miller, (1988) Computer Applic. Biol. Sci. 4:11-17, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has between 50-100% sequenceidentity, preferably at least 50% sequence identity, preferably at least60% sequence identity, preferably at least 70%, more preferably at least80%, more preferably at least 90% and most preferably at least 95%,compared to a reference sequence using one of the alignment programsdescribed using standard parameters. One of skill will recognize thatthese values can be appropriately adjusted to determine correspondingidentity of proteins encoded by two nucleotide sequences by taking intoaccount codon degeneracy, amino acid similarity, reading framepositioning and the like. Substantial identity of amino acid sequencesfor these purposes normally means sequence identity of between 55-100%,preferably at least 55%, preferably at least 60%, more preferably atleast 70%, 80%, 90% and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.The degeneracy of the genetic code allows for many amino acidssubstitutions that lead to variety in the nucleotide sequence that codefor the same amino acid, hence it is possible that the DNA sequencecould code for the same polypeptide but not hybridize to each otherunder stringent conditions. This may occur, e.g., when a copy of anucleic acid is created using the maximum codon degeneracy permitted bythe genetic code. One indication that two nucleic acid sequences aresubstantially identical is that the polypeptide, which the first nucleicacid encodes, is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

The terms “substantial identity” in the context of a peptide indicatesthat a peptide comprises a sequence with between 55-100% sequenceidentity to a reference sequence preferably at least 55% sequenceidentity, preferably 60% preferably 70%, more preferably 80%, mostpreferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Preferably, optimalalignment is conducted using the homology alignment algorithm ofNeedleman and Wunsch, supra. An indication that two peptide sequencesare substantially identical is that one peptide is immunologicallyreactive with antibodies raised against the second peptide. Thus, apeptide is substantially identical to a second peptide, for example,where the two peptides differ only by a conservative substitution. Inaddition, a peptide can be substantially identical to a second peptidewhen they differ by a non-conservative change if the epitope that theantibody recognizes is substantially identical. Peptides, which are“substantially similar” share sequences as, noted above except thatresidue positions, which are not identical, may differ by conservativeamino acid changes.

The invention discloses dimerization domain polynucleotides andpolypeptides. The novel nucleotides and proteins of the invention havean expression pattern which indicates that they alter cell wallformation and thus play an important role in plant development. Thepolynucleotides are expressed in various plant tissues. Thepolynucleotides and polypeptides thus provide an opportunity tomanipulate plant development to alter seed and vegetative tissuedevelopment, timing or composition. This may be used to create a sterileplant, a seedless plant or a plant with altered endosperm composition.

Nucleic Acids

The present invention provides, inter alia, isolated nucleic acids ofRNA, DNA and analogs and/or chimeras thereof, comprising a dimerizationdomain polynucleotide.

The present invention also includes polynucleotides optimized forexpression in different organisms. For example, for expression of thepolynucleotide in a maize plant, the sequence can be altered to accountfor specific codon preferences and to alter GC content as according toMurray, et al, supra. Maize codon usage for 28 genes from maize plantsis listed in Table 4 of Murray, et al., supra.

The dimerization domain nucleic acids include isolated dimerizationdomain polynucleotides which are inclusive of:

-   -   (a) a polynucleotide encoding a dimerization domain polypeptide        and conservatively modified and polymorphic variants thereof;    -   (b) a polynucleotide having at least 70% sequence identity with        polynucleotides of (a) or (b);    -   (c) complementary sequences of polynucleotides of (a) or (b).

The following table, Table 1, lists the specific identities of thesequences disclosed herein.

TABLE 1 SEQ ID NO: Identity SEQ ID NO: 1 MS-S2A promoter SEQ ID NO: 2ZmCesA10 polynucleotide SEQ ID NO: 3 Pin II terminator SEQ ID NO: 4 F3.7promoter SEQ ID NO: 5 ZmCesA4 polynucleotide SEQ ID NO: 6 ZmD8polynucleotide SEQ ID NO: 7 ZmNAS2 promoter SEQ ID NO: 8 ZmNAS2 5′UTRSEQ ID NO: 9 ZmD8 Dimerization Domain polynucletide (start and stopcodons are artificial appendages to the 243-331 coding sequence) SEQ IDNO: 10 NOS terminator SEQ ID NO: 11 ZmFTM1 polynucleotide SEQ ID NO: 12GmGAl1 polynucleotide SEQ ID NO: 13 ZRP2.47 promoter SEQ ID NO: 14 ADH1intron SEQ ID NO: 15 ZmRootMet2 promoter SEQ ID NO: 16 ZmCesA10polypeptide SEQ ID NO: 17 ZmCesA4 polypeptide SEQ ID NO: 18 ZmD8polypeptide SEQ ID NO: 19 ZmD8 243-331 Dimerization Domain polypeptide(ATG start codon is artificial and leads to an N-terminal methionineadded to the 243-331 amino acids). SEQ ID NO: 20 ZmFTM1 polypeptide SEQID NO: 21 GmGAl1 Dimerization Domain polypeptide SEQ ID NO: 22 GmGAl1polypeptide SEQ ID NO: 23 Gm 05g27190.1 SEQ ID NO: 24 Gm 05g27190.1Dimerization Domain SEQ ID NO: 25 Gm 08g10140.1 SEQ ID NO: 26 Gm08g10140.1 Dimerization Domain SEQ ID NO: 27 Gm 11g33720.1 SEQ ID NO: 28Gm 11g33720.1 Dimerization Domain SEQ ID NO: 29 Gm 18g04500.1 SEQ ID NO:30 Gm 18g04500.1 Dimerization Domain SEQ ID NO: 31 At GAl SEQ ID NO: 32At GAl Dimerization Domain SEQ ID NO: 33 At RGA SEQ ID NO: 34 At RGADimerization Domain SEQ ID NO: 35 At RGL1 SEQ ID NO: 36 At RGL1Dimerization Domain SEQ ID NO: 37 At RGL2 SEQ ID NO: 38 At RGL2Dimerization Domain SEQ ID NO: 39 At RGL3 SEQ ID NO: 40 At RGL3Dimerization Domain SEQ ID NO: 41 Consensus Dimerization Domain SEQ IDNO: 42 Primer SEQ ID NO: 43 Primer

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using(a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified or otherwise constructedfrom a fungus or bacteria.

The nucleic acids may conveniently comprise sequences in addition to apolynucleotide of the present invention. For example, a multi-cloningsite comprising one or more endonuclease restriction sites may beinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the polynucleotide sequence—isoptionally a vector, adapter or linker for cloning and/or expression ofa polynucleotide of the present invention. Additional sequences may beadded to such cloning and/or expression sequences to optimize theirfunction in cloning and/or expression, to aid in isolation of thepolynucleotide or to improve the introduction of the polynucleotide intoa cell. Typically, the length of a nucleic acid of the present inventionless the length of its polynucleotide of the present invention is lessthan 20 kilobase pairs, often less than 15 kb and frequently less than10 kb. Use of cloning vectors, expression vectors, adapters, and linkersis well known in the art. Exemplary nucleic acids include such vectorsas: M13, lambda ZAP Express, lambda ZAP II, lambda gt10, lambda gt11,pBK-CMV, pBK-RSV, pBluescript II, lambda DASH II, lambda EMBL 3, lambdaEMBL 4, pWE15, SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS+/−, pSG5, pBK,pCR-Script, pET, pSPUTK, p3′SS, pGEM, pSK+/−, pGEX, pSPORTI and II,pOPRSVI CAT, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pOG44, pOG45,pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405, pRS406, pRS413, pRS414,pRS415, pRS416, lambda MOSSlox and lambda MOSElox. Optional vectors forthe present invention, include but are not limited to, lambda ZAP II andpGEX. For a description of various nucleic acids see, e.g., StratageneCloning Systems, Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and,Amersham Life Sciences, Inc, Catalog '97 (Arlington Heights, Ill.).

Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be preparedby direct chemical synthesis by methods such as the phosphotriestermethod of Narang, et al., (1979) Meth. Enzymol. 68:90-9; thephosphodiester method of Brown, et al., (1979) Meth. Enzymol. 68:109-51;the diethylphosphoramidite method of Beaucage, et al., (1981) Tetra.Letts. 22(20):1859-62; the solid phase phosphoramidite triester methoddescribed by Beaucage, et al., supra, e.g., using an automatedsynthesizer, e.g., as described in Needham-VanDevanter, et al., (1984)Nucleic Acids Res. 12:6159-68 and the solid support method of U.S. Pat.No. 4,458,066. Chemical synthesis generally produces a single strandedoligonucleotide. This may be converted into double stranded DNA byhybridization with a complementary sequence or by polymerization with aDNA polymerase using the single strand as a template. One of skill willrecognize that while chemical synthesis of DNA is limited to sequencesof about 100 bases, longer sequences may be obtained by the ligation ofshorter sequences.

UTRs and Codon Preference

In general, translational efficiency has been found to be regulated byspecific sequence elements in the 5′ non-coding or untranslated region(5′ UTR) of the RNA. Positive sequence motifs include translationalinitiation consensus sequences (Kozak, (1987) Nucleic Acids Res.15:8125) and the 5<G> 7 methyl GpppG RNA cap structure (Drummond, etal., (1985) Nucleic Acids Res. 13:7375). Negative elements includestable intramolecular 5′ UTR stem-loop structures (Muesing, et al.,(1987) Cell 48:691) and AUG sequences or short open reading framespreceded by an appropriate AUG in the 5′ UTR (Kozak, supra, Rao, et al.,(1988) Mol. and Cell. Biol. 8:284). Accordingly, the present inventionprovides 5′ and/or 3′ UTR regions for modulation of translation ofheterologous coding sequences.

Further, the polypeptide-encoding segments of the polynucleotides of thepresent invention can be modified to alter codon usage. Altered codonusage can be employed to alter translational efficiency and/or tooptimize the coding sequence for expression in a desired host or tooptimize the codon usage in a heterologous sequence for expression inmaize. Codon usage in the coding regions of the polynucleotides of thepresent invention can be analyzed statistically using commerciallyavailable software packages such as “Codon Preference” available fromthe University of Wisconsin Genetics Computer Group. See, Devereaux, etal., (1984) Nucleic Acids Res. 12:387-395; or MacVector 4.1 (EastmanKodak Co., New Haven, Conn.). Thus, the present invention provides acodon usage frequency characteristic of the coding region of at leastone of the polynucleotides of the present invention. The number ofpolynucleotides (3 nucleotides per amino acid) that can be used todetermine a codon usage frequency can be any integer from 3 to thenumber of polynucleotides of the present invention as provided herein.Optionally, the polynucleotides will be full-length sequences. Anexemplary number of sequences for statistical analysis can be at least1, 5, 10, 20, 50 or 100.

Sequence Shuffling

The present invention provides methods for sequence shuffling usingpolynucleotides of the present invention, and compositions resultingtherefrom. Sequence shuffling is described in PCT publication number96/19256. See also, Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-9 and Zhao, et al., (1998) Nature Biotech 16:258-61. Generally,sequence shuffling provides a means for generating libraries ofpolynucleotides having a desired characteristic, which can be selectedor screened for. Libraries of recombinant polynucleotides are generatedfrom a population of related sequence polynucleotides, which comprisesequence regions, which have substantial sequence identity and can behomologously recombined in vitro or in vivo. The population ofsequence-recombined polynucleotides comprises a subpopulation ofpolynucleotides which possess desired or advantageous characteristicsand which can be selected by a suitable selection or screening method.The characteristics can be any property or attribute capable of beingselected for or detected in a screening system, and may includeproperties of: an encoded protein, a transcriptional element, a sequencecontrolling transcription, RNA processing, RNA stability, chromatinconformation, translation or other expression property of a gene ortransgene, a replicative element, a protein-binding element, or thelike, such as any feature which confers a selectable or detectableproperty. In some embodiments, the selected characteristic will be analtered K_(m) and/or K_(a), over the wild-type protein as providedherein. In other embodiments, a protein or polynucleotide generated fromsequence shuffling will have a ligand binding affinity greater than thenon-shuffled wild-type polynucleotide. In yet other embodiments, aprotein or polynucleotide generated from sequence shuffling will have analtered pH optimum as compared to the non-shuffled wild-typepolynucleotide. The increase in such properties can be at least 110%,120%, 130%, 140% or greater than 150% of the wild-type value.

Recombinant Expression Cassettes

The present invention further provides recombinant expression cassettescomprising a nucleic acid of the present invention. A nucleic acidsequence coding for the desired polynucleotide of the present invention,for example a cDNA or a genomic sequence encoding a polypeptide longenough to code for an active protein of the present invention, can beused to construct a recombinant expression cassette which can beintroduced into the desired host cell. A recombinant expression cassettewill typically comprise a polynucleotide of the present inventionoperably linked to transcriptional initiation regulatory sequences whichwill direct the transcription of the polynucleotide in the intended hostcell, such as tissues of a transformed plant.

For example, plant expression vectors may include (1) a cloned plantgene under the transcriptional control of 5′ and 3′ regulatory sequencesand (2) a dominant selectable marker. Such plant expression vectors mayalso contain, if desired, a promoter regulatory region (e.g., oneconferring inducible or constitutive, environmentally- ordevelopmentally-regulated or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site and/ora polyadenylation signal.

A plant promoter fragment can be employed which will direct expressionof a polynucleotide of the present invention in all tissues of aregenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumefaciens, the Smas promoter, the cinnamylalcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nospromoter, the rubisco promoter, the GRP1-8 promoter, the 35S promoterfrom cauliflower mosaic virus (CaMV), as described in Odell, et al.,(1985) Nature 313:810-2; rice actin (McElroy, et al., (1990) Plant Cell163-171); ubiquitin (Christensen, et al., (1992) Plant Mol. Biol.12:619-632 and Christensen, et al., (1992) Plant Mol. Biol. 18:675-89);pEMU (Last, et al., (1991) Theor. Appl. Genet. 81:581-8); MAS (Velten,et al., (1984) EMBO J. 3:2723-30) and maize H3 histone (Lepetit, et al.,(1992) Mol. Gen. Genet. 231:276-85 and Atanassvoa, et al., (1992) PlantJournal 2(3):291-300); ALS promoter, as described in PCT ApplicationNumber WO 96/30530; GOS2 (U.S. Pat. No. 6,504,083) and othertranscription initiation regions from various plant genes known to thoseof skill. For the present invention ubiquitin is the preferred promoterfor expression in monocot plants.

Alternatively, the plant promoter can direct expression of apolynucleotide of the present invention in a specific tissue or may beotherwise under more precise environmental or developmental control.Such promoters are referred to here as “inducible” promoters (Rab17,RAD29). Environmental conditions that may effect transcription byinducible promoters include pathogen attack, anaerobic conditions or thepresence of light. Examples of inducible promoters are the Adh1promoter, which is inducible by hypoxia or cold stress, the Hsp70promoter, which is inducible by heat stress and the PPDK promoter, whichis inducible by light.

Examples of promoters under developmental control include promoters thatinitiate transcription only, or preferentially, in certain tissues, suchas leaves, roots, fruit, seeds or flowers. The operation of a promotermay also vary depending on its location in the genome. Thus, aninducible promoter may become fully or partially constitutive in certainlocations.

If polypeptide expression is desired, it is generally desirable toinclude a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from a varietyof plant genes, or from T-DNA. The 3′ end sequence to be added can bederived from, for example, the nopaline synthase or octopine synthasegenes, or alternatively from another plant gene, or less preferably fromany other eukaryotic gene. Examples of such regulatory elements include,but are not limited to, 3′ termination and/or polyadenylation regionssuch as those of the Agrobacterium tumefaciens nopaline synthase (nos)gene (Bevan, et al., (1983) Nucleic Acids Res. 12:369-85); the potatoproteinase inhibitor II (PINII) gene (Keil, et al., (1986) Nucleic AcidsRes. 14:5641-50 and An, et al., (1989) Plant Cell 1:115-22) and the CaMV19S gene (Mogen, et al., (1990) Plant Cell 2:1261-72).

An intron sequence can be added to the 5′ untranslated region or thecoding sequence of the partial coding sequence to increase the amount ofthe mature message that accumulates in the cytosol. Inclusion of aspliceable intron in the transcription unit in both plant and animalexpression constructs has been shown to increase gene expression at boththe mRNA and protein levels up to 1000-fold (Buchman and Berg, (1988)Mol. Cell Biol. 8:4395-4405; Callis, et al., (1987) Genes Dev.1:1183-200). Such intron enhancement of gene expression is typicallygreatest when placed near the 5′ end of the transcription unit. Use ofmaize introns Adh1-S intron 1, 2 and 6, the Bronze-1 intron are known inthe art. See generally, THE MAIZE HANDBOOK, Chapter 116, Freeling andWalbot, eds., Springer, New York (1994).

Plant signal sequences, including, but not limited to, signal-peptideencoding DNA/RNA sequences which target proteins to the extracellularmatrix of the plant cell (Dratewka-Kos, et al., (1989) J. Biol. Chem.264:4896-900), such as the Nicotiana plumbaginifolia extension gene(DeLoose, et al., (1991) Gene 99:95-100); signal peptides which targetproteins to the vacuole, such as the sweet potato sporamin gene(Matsuka, et al., (1991) Proc. Natl. Acad. Sci. USA 88:834) and thebarley lectin gene (Wilkins, et al., (1990) Plant Cell, 2:301-13);signal peptides which cause proteins to be secreted, such as that ofPRIb (Lind, et al., (1992) Plant Mol. Biol. 18:47-53) or the barleyalpha amylase (BAA) (Rahmatullah, et al., (1989) Plant Mol. Biol.12:119, and hereby incorporated by reference) or signal peptides whichtarget proteins to the plastids such as that of rapeseed enoyl-Acpreductase (Verwaert, et al., (1994) Plant Mol. Biol. 26:189-202) areuseful in the invention. The barley alpha amylase signal sequence fusedto the dimerization domain component stack polynucleotide is thepreferred construct for expression in maize for the present invention.

The vector comprising the sequences from a polynucleotide of the presentdisclosure will typically comprise a marker gene, which confers aselectable phenotype on plant cells. Usually, the selectable marker genewill encode antibiotic resistance, with suitable genes including genescoding for resistance to the antibiotic spectinomycin (e.g., the aadagene), the streptomycin phosphotransferase (SPT) gene coding forstreptomycin resistance, the neomycin phosphotransferase (NPTII) geneencoding kanamycin or geneticin resistance, the hygromycinphosphotransferase (HPT) gene coding for hygromycin resistance, genescoding for resistance to herbicides which act to inhibit the action ofacetolactate synthase (ALS), in particular the sulfonylurea-typeherbicides (e.g., the acetolactate synthase (ALS) gene containingmutations leading to such resistance in particular the S4 and/or Hramutations), genes coding for resistance to herbicides which act toinhibit action of glutamine synthase, such as phosphinothricin or basta(e.g., the bar gene) or other such genes known in the art. The bar geneencodes resistance to the herbicide basta and the ALS gene encodesresistance to the herbicide chlorsulfuron.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of Agrobacterium tumefaciens described by Rogers, et al.,(1987) Meth. Enzymol. 153:253-77. These vectors are plant integratingvectors in that on transformation, the vectors integrate a portion ofvector DNA into the genome of the host plant. Exemplary A. tumefaciensvectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl, et al.,(1987) Gene 61:1-11 and Berger, et al., (1989) Proc. Natl. Acad. Sci.USA, 86:8402-6. Another useful vector herein is plasmid pBI101.2 that isavailable from CLONTECH Laboratories, Inc. (Palo Alto, Calif.).

Expression of Proteins in Host Cells

Using the nucleic acids of the present invention, one may express aprotein of the present invention in a recombinantly engineered cell suchas bacteria, yeast, insect, mammalian or preferably plant cells. Thecells produce the protein in a non-natural condition (e.g., in quantity,composition, location and/or time), because they have been geneticallyaltered through human intervention to do so.

It is expected that those of skill in the art are knowledgeable in thenumerous expression systems available for expression of a nucleic acidencoding a protein of the present invention. No attempt to describe indetail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

In brief summary, the expression of isolated nucleic acids encoding aprotein of the present invention will typically be achieved by operablylinking, for example, the DNA or cDNA to a promoter (which is eitherconstitutive or inducible), followed by incorporation into an expressionvector. The vectors can be suitable for replication and integration ineither prokaryotes or eukaryotes. Typical expression vectors containtranscription and translation terminators, initiation sequences andpromoters useful for regulation of the expression of the DNA encoding aprotein of the present invention. To obtain high level expression of acloned gene, it is desirable to construct expression vectors whichcontain, at the minimum, a strong promoter, such as ubiquitin, to directtranscription, a ribosome binding site for translational initiation anda transcription/translation terminator. Constitutive promoters areclassified as providing for a range of constitutive expression. Thus,some are weak constitutive promoters and others are strong constitutivepromoters. Generally, by “weak promoter” is intended a promoter thatdrives expression of a coding sequence at a low level. By “low level” isintended at levels of about 1/10,000 transcripts to about 1/100,000transcripts to about 1/500,000 transcripts. Conversely, a “strongpromoter” drives expression of a coding sequence at a “high level” orabout 1/10 transcripts to about 1/100 transcripts to about 1/1,000transcripts.

One of skill would recognize that modifications could be made to aprotein of the present invention without diminishing its biologicalactivity. Some modifications may be made to facilitate the cloning,expression or incorporation of the targeting molecule into a fusionprotein. Such modifications are well known to those of skill in the artand include, for example, a methionine added at the amino terminus toprovide an initiation site or additional amino acids (e.g., poly His)placed on either terminus to create conveniently located restrictionsites or termination codons or purification sequences.

Expression in Prokaryotes

Prokaryotic cells may be used as hosts for expression. Prokaryotes mostfrequently are represented by various strains of E. coli; however, othermicrobial strains may also be used. Commonly used prokaryotic controlsequences which are defined herein to include promoters fortranscription initiation, optionally with an operator, along withribosome binding site sequences, include such commonly used promoters asthe beta lactamase (penicillinase) and lactose (lac) promoter systems(Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promotersystem (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057) and thelambda derived P L promoter and N-gene ribosome binding site (Shimatake,et al., (1981) Nature 292:128). The inclusion of selection markers inDNA vectors transfected in E. coli is also useful. Examples of suchmarkers include genes specifying resistance to ampicillin, tetracyclineor chloramphenicol.

The vector is selected to allow introduction of the gene of interestinto the appropriate host cell. Bacterial vectors are typically ofplasmid or phage origin. Appropriate bacterial cells are infected withphage vector particles or transfected with naked phage vector DNA. If aplasmid vector is used, the bacterial cells are transfected with theplasmid vector DNA. Expression systems for expressing a protein of thepresent invention are available using Bacillus sp. and Salmonella(Palva, et al., (1983) Gene 22:229-35; Mosbach, et al., (1983) Nature302:543-5). The pGEX-4T-1 plasmid vector from Pharmacia is the preferredE. coli expression vector for the present invention.

Expression in Eukaryotes

A variety of eukaryotic expression systems such as yeast, insect celllines, plant and mammalian cells, are known to those of skill in theart. As explained briefly below, the present invention can be expressedin these eukaryotic systems. In some embodiments,transformed/transfected plant cells, as discussed infra, are employed asexpression systems for production of the proteins of the instantinvention.

Synthesis of heterologous proteins in yeast is well known. Sherman, etal., (1982) METHODS IN YEAST GENETICS, Cold Spring Harbor Laboratory isa well recognized work describing the various methods available toproduce the protein in yeast. Two widely utilized yeasts for productionof eukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.Vectors, strains and protocols for expression in Saccharomyces andPichia are known in the art and available from commercial suppliers(e.g., Invitrogen). Suitable vectors usually have expression controlsequences, such as promoters, including 3-phosphoglycerate kinase oralcohol oxidase, and an origin of replication, termination sequences andthe like as desired.

A protein of the present invention, once expressed, can be isolated fromyeast by lysing the cells and applying standard protein isolationtechniques to the lysates or the pellets. The monitoring of thepurification process can be accomplished by using Western blottechniques or radioimmunoassay of other standard immunoassay techniques.

The sequences encoding proteins of the present invention can also beligated to various expression vectors for use in transfecting cellcultures of, for instance, mammalian, insect or plant origin. Mammaliancell systems often will be in the form of monolayers of cells althoughmammalian cell suspensions may also be used. A number of suitable hostcell lines capable of expressing intact proteins have been developed inthe art, and include the HEK293, BHK21 and CHO cell lines. Expressionvectors for these cells can include expression control sequences, suchas an origin of replication, a promoter (e.g., the CMV promoter, a HSVtk promoter or pgk (phosphoglycerate kinase) promoter), an enhancer(Queen, et al., (1986) Immunol. Rev. 89:49) and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites (e.g., an SV40 large T Ag poly A addition site)and transcriptional terminator sequences. Other animal cells useful forproduction of proteins of the present invention are available, forinstance, from the American Type Culture Collection Catalogue of CellLines and Hybridomas (7^(th) ed., 1992).

Appropriate vectors for expressing proteins of the present invention ininsect cells are usually derived from the SF9 baculovirus. Suitableinsect cell lines include mosquito larvae, silkworm, armyworm, moth andDrosophila cell lines such as a Schneider cell line (see, e.g.,Schneider, (1987) J. Embryol. Exp. Morphol. 27:353-65).

As with yeast, when higher animal or plant host cells are employed,polyadenlyation or transcription terminator sequences are typicallyincorporated into the vector. An example of a terminator sequence is thepolyadenlyation sequence from the bovine growth hormone gene. Sequencesfor accurate splicing of the transcript may also be included. An exampleof a splicing sequence is the VP1 intron from SV40 (Sprague, et al.,(1983) J. Virol. 45:773-81). Additionally, gene sequences to controlreplication in the host cell may be incorporated into the vector such asthose found in bovine papilloma virus type-vectors (Saveria-Campo,“Bovine Papilloma Virus DNA a Eukaryotic Cloning Vector,” in DNACLONING: A PRACTICAL APPROACH, vol. II, Glover, ed., IRL Press,Arlington, Va., pp. 213-38 (1985)).

In addition, the gene for dimerization domain placed in the appropriateplant expression vector can be used to transform plant cells. Thepolypeptide can then be isolated from plant callus or the transformedcells can be used to regenerate transgenic plants. Such transgenicplants can be harvested and the appropriate tissues (seed or leaves, forexample) can be subjected to large scale protein extraction andpurification techniques.

Plant Transformation Methods

Numerous methods for introducing foreign genes into plants are known andcan be used to insert a dimerization domain polynucleotide into a planthost, including biological and physical plant transformation protocols.See, e.g., Miki, et al., “Procedure for Introducing Foreign DNA intoPlants,” in METHODS IN PLANT MOLECULAR BIOLOGY AND BIOTECHNOLOGY, Glickand Thompson, eds., CRC Press, Inc., Boca Raton, pp. 67-88 (1993). Themethods chosen vary with the host plant, and include chemicaltransfection methods such as calcium phosphate, microorganism-mediatedgene transfer such as Agrobacterium (Horsch, et al., (1985) Science227:1229-31), electroporation, micro-injection and biolisticbombardment.

Expression cassettes and vectors and in vitro culture methods for plantcell or tissue transformation and regeneration of plants are known andavailable. See, e.g., Gruber, et al., “Vectors for PlantTransformation,” in METHODS IN PLANT MOLECULAR BIOLOGY ANDBIOTECHNOLOGY, supra, pp. 89-119.

The isolated polynucleotides or polypeptides may be introduced into theplant by one or more techniques typically used for direct delivery intocells. Such protocols may vary depending on the type of organism, cell,plant or plant cell, i.e., monocot or dicot, targeted for genemodification. Suitable methods of transforming plant cells includemicroinjection (Crossway, et al., (1986) Biotechniques 4:320-334; andU.S. Pat. No. 6,300,543), electroporation (Riggs, et al., (1986) Proc.Natl. Acad. Sci. USA 83:5602-5606), direct gene transfer (Paszkowski, etal., (1984) EMBO J. 3:2717-2722) and ballistic particle acceleration(see, for example, Sanford, et al., U.S. Pat. No. 4,945,050; WO 91/10725and McCabe, et al., (1988) Biotechnology 6:923-926). Also see, Tomes, etal., Direct DNA Transfer into Intact Plant Cells Via MicroprojectileBombardment pp. 197-213 in Plant Cell, Tissue and Organ Culture,Fundamental Methods eds. Gamborg and Phillips, Springer-Verlag BerlinHeidelberg New York, 1995; U.S. Pat. No. 5,736,369 (meristem);Weissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al.,(1987) Particulate Science and Technology 5:27-37 (onion); Christou, etal., (1988) Plant Physiol. 87:671-674 (soybean); Datta, et al., (1990)Biotechnology 8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad.Sci. USA 85:4305-4309 (maize); Klein, et al., (1988) Biotechnology6:559-563 (maize); WO 91/10725 (maize); Klein, et al., (1988) PlantPhysiol. 91:440-444 (maize); Fromm, et al., (1990) Biotechnology8:833-839 and Gordon-Kamm, et al., (1990) Plant Cell 2:603-618 (maize);Hooydaas-Van Slogteren and Hooykaas, (1984) Nature (London) 311:763-764;Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) In The Experimental Manipulation ofOvule Tissues, ed. G. P. Chapman, et al., pp. 197-209; Longman, N.Y.(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); U.S. Pat. No. 5,693,512 (sonication);D'Halluin, et al., (1992) Plant Cell 4:1495-1505 (electroporation); Li,et al., (1993) Plant Cell Reports 12:250-255 and Christou and Ford,(1995) Annals of Botany 75:407-413 (rice); Osjoda, et al., (1996) NatureBiotech. 14:745-750; Agrobacterium mediated maize transformation (U.S.Pat. No. 5,981,840); silicon carbide whisker methods (Frame, et al.,(1994) Plant J. 6:941-948); laser methods (Guo, et al., (1995)Physiologia Plantarum 93:19-24); sonication methods (Bao, et al., (1997)Ultrasound in Medicine & Biology 23:953-959; Finer and Finer, (2000)Lett Appl Microbiol. 30:406-10; Amoah, et al., (2001) J Exp Bot52:1135-42); polyethylene glycol methods (Krens, et al., (1982) Nature296:72-77); protoplasts of monocot and dicot cells can be transformedusing electroporation (Fromm, et al., (1985) Proc. Natl. Acad. Sci. USA82:5824-5828) and microinjection (Crossway, et al., (1986) Mol. Gen.Genet. 202:179-185), all of which are herein incorporated by reference.

Agrobacterium-Mediated Transformation

The most widely utilized method for introducing an expression vectorinto plants is based on the natural transformation system ofAgrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria, which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectively, carry genesresponsible for genetic transformation of plants. See, e.g., Kado,(1991) Crit. Rev. Plant Sci. 10:1. Descriptions of the Agrobacteriumvector systems and methods for Agrobacterium-mediated gene transfer areprovided in Gruber, et al., supra; Miki, et al., supra and Moloney, etal., (1989) Plant Cell Reports 8:238.

Similarly, the gene can be inserted into the T-DNA region of a Ti or Riplasmid derived from A. tumefaciens or A. rhizogenes, respectively.Thus, expression cassettes can be constructed as above, using theseplasmids. Many control sequences are known which when coupled to aheterologous coding sequence and transformed into a host organism showfidelity in gene expression with respect to tissue/organ specificity ofthe original coding sequence. See, e.g., Benfey and Chua, (1989) Science244:174-81. Particularly suitable control sequences for use in theseplasmids are promoters for constitutive leaf-specific expression of thegene in the various target plants. Other useful control sequencesinclude a promoter and terminator from the nopaline synthase gene (NOS).The NOS promoter and terminator are present in the plasmid pARC2,available from the American Type Culture Collection and designated ATCC67238. If such a system is used, the virulence (vir) gene from eitherthe Ti or Ri plasmid must also be present, either along with the T-DNAportion or via a binary system where the vir gene is present on aseparate vector. Such systems, vectors for use therein, and methods oftransforming plant cells are described in U.S. Pat. No. 4,658,082; USPatent Application Serial Number 913,914, filed Oct. 1, 1986, asreferenced in U.S. Pat. No. 5,262,306, issued Nov. 16, 1993 and Simpson,et al., (1986) Plant Mol. Biol. 6:403-15 (also referenced in the '306patent), all incorporated by reference in their entirety.

Once constructed, these plasmids can be placed into A. rhizogenes or A.tumefaciens and these vectors used to transform cells of plant species,which are ordinarily susceptible to Fusarium or Alternaria infection.Several other transgenic plants are also contemplated by the presentinvention including but not limited to soybean, corn, sorghum, alfalfa,rice, clover, cabbage, banana, coffee, celery, tobacco, cowpea, cotton,melon and pepper. The selection of either A. tumefaciens or A.rhizogenes will depend on the plant being transformed thereby. Ingeneral A. tumefaciens is the preferred organism for transformation.Most dicotyledonous plants, some gymnosperms and a few monocotyledonousplants (e.g., certain members of the Liliales and Arales) aresusceptible to infection with A. tumefaciens. A. rhizogenes also has awide host range, embracing most dicots and some gymnosperms, whichincludes members of the Leguminosae, Compositae and Chenopodiaceae.Monocot plants can now be transformed with some success. EP ApplicationNumber 604 662 A1 discloses a method for transforming monocots usingAgrobacterium. EP Application Number 672 752 A1 discloses a method fortransforming monocots with Agrobacterium using the scutellum of immatureembryos. Ishida, et al., discuss a method for transforming maize byexposing immature embryos to A. tumefaciens (Nature Biotechnology14:745-50 (1996)).

Once transformed, these cells can be used to regenerate transgenicplants. For example, whole plants can be infected with these vectors bywounding the plant and then introducing the vector into the wound site.Any part of the plant can be wounded, including leaves, stems and roots.Alternatively, plant tissue, in the form of an explant, such ascotyledonary tissue or leaf disks, can be inoculated with these vectors,and cultured under conditions, which promote plant regeneration. Rootsor shoots transformed by inoculation of plant tissue with A. rhizogenesor A. tumefaciens, containing the gene coding for the fumonisindegradation enzyme, can be used as a source of plant tissue toregenerate fumonisin-resistant transgenic plants, either via somaticembryogenesis or organogenesis. Examples of such methods forregenerating plant tissue are disclosed in Shahin, (1985) Theor. Appl.Genet. 69:235-40; U.S. Pat. No. 4,658,082; Simpson, et al., supra and USPatent Application Serial Numbers 913,913 and 913,914, both filed Oct.1, 1986, as referenced in U.S. Pat. No. 5,262,306, issued Nov. 16, 1993,the entire disclosures therein incorporated herein by reference.

Direct Gene Transfer

Despite the fact that the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice (Hiei, et al.,(1994) The Plant Journal 6:271-82). Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation, where DNA is carried on thesurface of microprojectiles measuring about 1 to 4 μm. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate the plant cell walls and membranes (Sanford, etal., (1987) Part. Sci. Technol. 5:27; Sanford, (1988) Trends Biotech6:299; Sanford, (1990) Physiol. Plant 79:206 and Klein, et al., (1992)Biotechnology 10:268).

Another method for physical delivery of DNA to plants is sonication oftarget cells as described in Zang, et al., (1991) BioTechnology 9:996.Alternatively, liposome or spheroplast fusions have been used tointroduce expression vectors into plants. See, e.g., Deshayes, et al.,(1985) EMBO J. 4:2731 and Christou, et al., (1987) Proc. Natl. Acad.Sci. USA 84:3962. Direct uptake of DNA into protoplasts using CaCl₂precipitation, polyvinyl alcohol, or poly-L-ornithine has also beenreported. See, e.g., Hain, et al., (1985) Mol. Gen. Genet. 199:161 andDraper, et al., (1982) Plant Cell Physiol. 23:451.

Electroporation of protoplasts and whole cells and tissues has also beendescribed. See, e.g., Donn, et al., (1990) in Abstracts of the VIlthIntl. Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p. 53;D'Halluin, et al., (1992) Plant Cell 4:1495-505 and Spencer, et al.,(1994) Plant Mol. Biol. 24:51-61.

Increasing the Activity and/or Level of a Dimerization DomainPolypeptide

Methods are provided to increase the activity and/or level of thedimerization domain polypeptide. An increase in the level and/oractivity of the dimerization domain polypeptide can be achieved byproviding to the plant a dimerization domain polypeptide. Thedimerization domain polypeptide can be provided by introducing the aminoacid sequence encoding the dimerization domain polypeptide into theplant, introducing into the plant a nucleotide sequence encoding adimerization domain polypeptide or alternatively by selecting fordifferent variants of the genomic locus encoding the dimerization domainpolypeptide of the invention.

As discussed elsewhere herein, many methods are known in the art forproviding a polypeptide to a plant including, but not limited to, directintroduction of the polypeptide into the plant, introducing into theplant (transiently or stably) a polynucleotide construct encoding apolypeptide having dimerization domain component stack which directsplant development activity. It is also recognized that the methods ofthe invention may employ a polynucleotide that is not capable ofdirecting, in the transformed plant, the expression of a protein or anRNA. Thus, the level and/or activity of a dimerization domainpolypeptide may be increased by altering the gene encoding thedimerization domain polypeptide or its promoter. See, e.g., Kmiec, U.S.Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868. Thereforemutagenized plants that carry mutations in dimerization domain genes,where the mutations increase expression of the dimerization domain geneor increase the plant growth and/or dimerization domain activity of theencoded dimerization domain polypeptide are provided.

Reducing the Activity and/or Level of a Dimerization Domain Polypeptide

Methods are provided to reduce or eliminate the activity of adimerization domain polypeptide of the invention by transforming a plantcell with an expression cassette that expresses a polynucleotide thatinhibits the expression of the dimerization domain polypeptide. Thepolynucleotide may inhibit the expression of the dimerization domainpolypeptide directly, by preventing translation of the dimerizationdomain messenger RNA, or indirectly, by encoding a polypeptide thatinhibits the transcription or translation of a dimerization domain geneencoding a dimerization domain polypeptide. Methods for inhibiting oreliminating the expression of a gene in a plant are well known in theart, and any such method may be used in the present invention to inhibitthe expression of a dimerization domain polypeptide.

The expression of a target polypeptide is inhibited if the protein levelof the polypeptide is less than 70% of the protein level of thepolypeptide in a plant that has not been genetically modified ormutagenized to inhibit the expression of that dimerization domainpolypeptide. In particular embodiments of the invention, the proteinlevel of the dimerization domain polypeptide in a modified plantaccording to the invention is less than 60%, less than 50%, less than40%, less than 30%, less than 20%, less than 10%, less than 5% or lessthan 2% of the protein level of the same dimerization domain polypeptidein a plant that is not a mutant or that has not been geneticallymodified to inhibit the expression of that dimerization domainpolypeptide. The expression level of the dimerization domain polypeptidemay be measured directly, for example, by assaying for the level ofdimerization domain polypeptide expressed in the plant cell or plant, orindirectly, for example, by measuring the plant growth and/ordimerization domain activity of the dimerization domain polypeptide inthe plant cell or plant, or by measuring the biomass in the plant.Methods for performing such assays are described elsewhere herein.

In other embodiments of the invention, the activity of the dimerizationdomain polypeptides is reduced or eliminated by transforming a plantcell with an expression cassette comprising a polynucleotide encoding apolypeptide that inhibits the activity of a dimerization domainpolypeptide. The plant growth and/or dimerization domain activity of adimerization domain component stack polypeptide is inhibited accordingto the present invention if the plant growth and/or dimerization domainactivity of the dimerization domain component stack polypeptide is lessthan 70% of the plant growth and/or dimerization domain activity of thesame dimerization domain polypeptide in a plant that has not beenmodified to inhibit the plant growth and/or dimerization domain activityof that dimerization domain component stack polypeptide. In particularembodiments of the invention, the plant growth and/or dimerizationdomain activity of the dimerization domain polypeptide in a modifiedplant according to the invention is less than 60%, less than 50%, lessthan 40%, less than 30%, less than 20%, less than 10% or less than 5% ofthe plant growth and/or dimerization domain activity of the samedimerization domain polypeptide in a plant that that has not beenmodified to inhibit the expression of that dimerization domainpolypeptide. The plant growth and/or dimerization domain activity of adimerization domain polypeptide is “eliminated” according to theinvention when it is not detectable by the assay methods describedelsewhere herein. Methods of determining the plant growth and/ordimerization domain activity of a dimerization domain polypeptide aredescribed elsewhere herein.

In other embodiments, the activity of a dimerization domain componetstack polypeptide may be reduced or eliminated by disrupting the geneencoding the dimerization domain polypeptide. The invention encompassesmutagenized plants that carry mutations in dimerization domain genes,where the mutations reduce expression of the dimerization domain gene orinhibit the plant growth and/or dimerization domain activity of theencoded dimerization domain polypeptide.

Thus, many methods may be used to reduce or eliminate the activity of adimerization domain polypeptide. In addition, more than one method maybe used to reduce the activity of a single dimerization domainpolypeptide. Non-limiting examples of methods of reducing or eliminatingthe expression of dimerization domain polypeptides are given below.

1. Polynucleotide-Based Methods:

In some embodiments of the present invention, a plant is transformedwith an expression cassette that is capable of expressing apolynucleotide that inhibits the expression of a dimerization domainpolypeptide of the invention. The term “expression” as used hereinrefers to the biosynthesis of a gene product, including thetranscription and/or translation of said gene product. For example, forthe purposes of the present invention, an expression cassette capable ofexpressing a polynucleotide that inhibits the expression of at least onedimerization domain polypeptide is an expression cassette capable ofproducing an RNA molecule that inhibits the transcription and/ortranslation of at least one dimerization domain polypeptide of theinvention. The “expression” or “production” of a protein or polypeptidefrom a DNA molecule refers to the transcription and translation of thecoding sequence to produce the protein or polypeptide, while the“expression” or “production” of a protein or polypeptide from an RNAmolecule refers to the translation of the RNA coding sequence to producethe protein or polypeptide.

Examples of polynucleotides that inhibit the expression of adimerization domain polypeptide are given below.

i. Sense Suppression/Cosuppression

In some embodiments of the invention, inhibition of the expression of adimerization domain polypeptide may be obtained by sense suppression orcosuppression. For cosuppression, an expression cassette is designed toexpress an RNA molecule corresponding to all or part of a messenger RNAencoding a dimerization domain polypeptide in the “sense” orientation.Over expression of the RNA molecule can result in reduced expression ofthe native gene. Accordingly, multiple plant lines transformed with thecosuppression expression cassette are screened to identify those thatshow the greatest inhibition of dimerization domain polypeptideexpression.

The polynucleotide used for cosuppression may correspond to all or partof the sequence encoding the dimerization domain polypeptide, all orpart of the 5′ and/or 3′ untranslated region of a dimerization domainpolypeptide transcript or all or part of both the coding sequence andthe untranslated regions of a transcript encoding a dimerization domainpolypeptide. In some embodiments where the polynucleotide comprises allor part of the coding region for the dimerization domain polypeptide,the expression cassette is designed to eliminate the start codon of thepolynucleotide so that no protein product will be translated.

Cosuppression may be used to inhibit the expression of plant genes toproduce plants having undetectable protein levels for the proteinsencoded by these genes. See, for example, Broin, et al., (2002) PlantCell 14:1417-1432. Cosuppression may also be used to inhibit theexpression of multiple proteins in the same plant. See, for example,U.S. Pat. No. 5,942,657. Methods for using cosuppression to inhibit theexpression of endogenous genes in plants are described in Flavell, etal., (1994) Proc. Natl. Acad. Sci. USA 91:3490-3496; Jorgensen, et al.,(1996) Plant Mol. Biol. 31:957-973; Johansen and Carrington, (2001)Plant Physiol. 126:930-938; Broin, et al., (2002) Plant Cell14:1417-1432; Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731;Yu, et al., (2003) Phytochemistry 63:753-763 and U.S. Pat. Nos.5,034,323, 5,283,184 and 5,942,657, each of which is herein incorporatedby reference. The efficiency of cosuppression may be increased byincluding a poly-dT region in the expression cassette at a position 3′to the sense sequence and 5′ of the polyadenylation signal. See, USPatent Application Publication Number 2002/0048814, herein incorporatedby reference. Typically, such a nucleotide sequence has substantialsequence identity to the sequence of the transcript of the endogenousgene, optimally greater than about 65% sequence identity, more optimallygreater than about 85% sequence identity, most optimally greater thanabout 95% sequence identity. See, U.S. Pat. Nos. 5,283,184 and5,034,323, herein incorporated by reference.

ii. Antisense Suppression

In some embodiments of the invention, inhibition of the expression ofthe dimerization domain polypeptide may be obtained by antisensesuppression. For antisense suppression, the expression cassette isdesigned to express an RNA molecule complementary to all or part of amessenger RNA encoding the dimerization domain polypeptide. Overexpression of the antisense RNA molecule can result in reducedexpression of the native gene. Accordingly, multiple plant linestransformed with the antisense suppression expression cassette arescreened to identify those that show the greatest inhibition ofdimerization domain polypeptide expression.

The polynucleotide for use in antisense suppression may correspond toall or part of the complement of the sequence encoding the dimerizationdomain polypeptide, all or part of the complement of the 5′ and/or 3′untranslated region of the dimerization domain transcript or all or partof the complement of both the coding sequence and the untranslatedregions of a transcript encoding the dimerization domain polypeptide. Inaddition, the antisense polynucleotide may be fully complementary (i.e.,100% identical to the complement of the target sequence) or partiallycomplementary (i.e., less than 100% identical to the complement of thetarget sequence) to the target sequence. Antisense suppression may beused to inhibit the expression of multiple proteins in the same plant.See, for example, U.S. Pat. No. 5,942,657. Furthermore, portions of theantisense nucleotides may be used to disrupt the expression of thetarget gene. Generally, sequences of at least 50 nucleotides, 100nucleotides, 200 nucleotides, 300, 400, 450, 500, 550 or greater may beused. Methods for using antisense suppression to inhibit the expressionof endogenous genes in plants are described, for example, in Liu, etal., (2002) Plant Physiol. 129:1732-1743 and U.S. Pat. Nos. 5,759,829and 5,942,657, each of which is herein incorporated by reference.Efficiency of antisense suppression may be increased by including apoly-dT region in the expression cassette at a position 3′ to theantisense sequence and 5′ of the polyadenylation signal. See, US PatentApplication Publication Number 2002/0048814, herein incorporated byreference.

iii. Double-Stranded RNA Interference

In some embodiments of the invention, inhibition of the expression of adimerization domain polypeptide may be obtained by double-stranded RNA(dsRNA) interference. For dsRNA interference, a sense RNA molecule likethat described above for cosuppression and an antisense RNA moleculethat is fully or partially complementary to the sense RNA molecule areexpressed in the same cell, resulting in inhibition of the expression ofthe corresponding endogenous messenger RNA.

Expression of the sense and antisense molecules can be accomplished bydesigning the expression cassette to comprise both a sense sequence andan antisense sequence. Alternatively, separate expression cassettes maybe used for the sense and antisense sequences. Multiple plant linestransformed with the dsRNA interference expression cassette orexpression cassettes are then screened to identify plant lines that showthe greatest inhibition of dimerization domain polypeptide expression.Methods for using dsRNA interference to inhibit the expression ofendogenous plant genes are described in Waterhouse, et al., (1998) Proc.Natl. Acad. Sci. USA 95:13959-13964, Liu, et al., (2002) Plant Physiol.129:1732-1743 and WO 99/49029, WO 99/53050, WO 99/61631 and WO 00/49035,each of which is herein incorporated by reference.

iv. Hairpin RNA Interference and Intron-Containing Hairpin RNAInterference

In some embodiments of the invention, inhibition of the expression ofone or a dimerization domain polypeptide may be obtained by hairpin RNA(hpRNA) interference or intron-containing hairpin RNA (ihpRNA)interference. These methods are highly efficient at inhibiting theexpression of endogenous genes. See, Waterhouse and Helliwell, (2003)Nat. Rev. Genet. 4:29-38 and the references cited therein.

For hpRNA interference, the expression cassette is designed to expressan RNA molecule that hybridizes with itself to form a hairpin structurethat comprises a single-stranded loop region and a base-paired stem. Thebase-paired stem region comprises a sense sequence corresponding to allor part of the endogenous messenger RNA encoding the gene whoseexpression is to be inhibited and an antisense sequence that is fully orpartially complementary to the sense sequence. Thus, the base-pairedstem region of the molecule generally determines the specificity of theRNA interference. hpRNA molecules are highly efficient at inhibiting theexpression of endogenous genes and the RNA interference they induce isinherited by subsequent generations of plants. See, for example, Chuangand Meyerowitz, (2000) Proc. Natl. Acad. Sci. USA 97:4985-4990;Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731 and Waterhouseand Helliwell, (2003) Nat. Rev. Genet. 4:29-38. Methods for using hpRNAinterference to inhibit or silence the expression of genes aredescribed, for example, in Chuang and Meyerowitz, (2000) Proc. Natl.Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol.129:1723-1731; Waterhouse and Helliwell, (2003) Nat. Rev. Genet.4:29-38; Pandolfini, et al., BMC Biotechnology 3:7 and US PatentApplication Publication Number 2003/0175965, each of which is hereinincorporated by reference. A transient assay for the efficiency of hpRNAconstructs to silence gene expression in vivo has been described byPanstruga, et al., (2003) Mol. Biol. Rep. 30:135-140, hereinincorporated by reference.

For ihpRNA, the interfering molecules have the same general structure asfor hpRNA, but the RNA molecule additionally comprises an intron that iscapable of being spliced in the cell in which the ihpRNA is expressed.The use of an intron minimizes the size of the loop in the hairpin RNAmolecule following splicing, and this increases the efficiency ofinterference. See, for example, Smith, et al., (2000) Nature407:319-320. In fact, Smith, et al., show 100% suppression of endogenousgene expression using ihpRNA-mediated interference. Methods for usingihpRNA interference to inhibit the expression of endogenous plant genesare described, for example, in Smith, et al., (2000) Nature 407:319-320;Wesley, et al., (2001) Plant J. 27:581-590; Wang and Waterhouse, (2001)Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell, (2003) Nat.Rev. Genet. 4:29-38; Helliwell and Waterhouse, (2003) Methods 30:289-295and US Patent Application Publication Number 2003/0180945, each of whichis herein incorporated by reference.

The expression cassette for hpRNA interference may also be designed suchthat the sense sequence and the antisense sequence do not correspond toan endogenous RNA. In this embodiment, the sense and antisense sequenceflank a loop sequence that comprises a nucleotide sequence correspondingto all or part of the endogenous messenger RNA of the target gene. Thus,it is the loop region that determines the specificity of the RNAinterference. See, for example, WO 02/00904, herein incorporated byreference.

v. Amplicon-Mediated Interference

Amplicon expression cassettes comprise a plant virus-derived sequencethat contains all or part of the target gene but generally not all ofthe genes of the native virus. The viral sequences present in thetranscription product of the expression cassette allow the transcriptionproduct to direct its own replication. The transcripts produced by theamplicon may be either sense or antisense relative to the targetsequence (i.e., the messenger RNA for the dimerization domainpolypeptide). Methods of using amplicons to inhibit the expression ofendogenous plant genes are described, for example, in Angell andBaulcombe, (1997) EMBO J. 16:3675-3684, Angell and Baulcombe, (1999)Plant J. 20:357-362 and U.S. Pat. No. 6,646,805, each of which is hereinincorporated by reference.

vi. Ribozymes

In some embodiments, the polynucleotide expressed by the expressioncassette of the invention is catalytic RNA or has ribozyme activityspecific for the messenger RNA of the dimerization domain polypeptide.Thus, the polynucleotide causes the degradation of the endogenousmessenger RNA, resulting in reduced expression of the dimerizationdomain polypeptide. This method is described, for example, in U.S. Pat.No. 4,987,071, herein incorporated by reference.

vii. Small Interfering RNA or Micro RNA

In some embodiments of the invention, inhibition of the expression of adimerization domain polypeptide may be obtained by RNA interference byexpression of a gene encoding a micro RNA (miRNA). miRNAs are regulatoryagents consisting of about 22 ribonucleotides. miRNA are highlyefficient at inhibiting the expression of endogenous genes. See, forexample, Javier, et al., (2003) Nature 425:257-263, herein incorporatedby reference.

For miRNA interference, the expression cassette is designed to expressan RNA molecule that is modeled on an endogenous miRNA gene. The miRNAgene encodes an RNA that forms a hairpin structure containing a22-nucleotide sequence that is complementary to another endogenous gene(target sequence). For suppression of dimerization domain expression,the 22-nucleotide sequence is selected from a dimerization domaintranscript sequence and contains 22 nucleotide of said dimerizationdomain sequence in sense orientation and 21 nucleotides of acorresponding antisense sequence that is complementary to the sensesequence. miRNA molecules are highly efficient at inhibiting theexpression of endogenous genes, and the RNA interference they induce isinherited by subsequent generations of plants.

2. Polypeptide-Based Inhibition of Gene Expression

In one embodiment, the polynucleotide encodes a zinc finger protein thatbinds to a gene encoding a dimerization domain polypeptide, resulting inreduced expression of the gene. In particular embodiments, the zincfinger protein binds to a regulatory region of a dimerization domaingene. In other embodiments, the zinc finger protein binds to a messengerRNA encoding a dimerization domain polypeptide and prevents itstranslation. Methods of selecting sites for targeting by zinc fingerproteins have been described, for example, in U.S. Pat. No. 6,453,242and methods for using zinc finger proteins to inhibit the expression ofgenes in plants are described, for example, in US Patent ApplicationPublication Number 2003/0037355, each of which is herein incorporated byreference.

3. Polypeptide-Based Inhibition of Protein Activity

In some embodiments of the invention, the polynucleotide encodes anantibody that binds to at least one dimerization domain polypeptide andreduces the dimerization domain activity of the dimerization domainpolypeptide. In another embodiment, the binding of the antibody resultsin increased turnover of the antibody-dimerization domain complex bycellular quality control mechanisms. The expression of antibodies inplant cells and the inhibition of molecular pathways by expression andbinding of antibodies to proteins in plant cells are well known in theart. See, for example, Conrad and Sonnewald, (2003) Nature Biotech.21:35-36, incorporated herein by reference.

4. Gene Disruption

In some embodiments of the present invention, the activity of adimerization domain polypeptide is reduced or eliminated by disruptingthe gene encoding the dimerization domain polypeptide. The gene encodingthe dimerization domain polypeptide may be disrupted by any method knownin the art. For example, in one embodiment, the gene is disrupted bytransposon tagging. In another embodiment, the gene is disrupted bymutagenizing plants using random or targeted mutagenesis and selectingfor plants that have reduced dimerization domain activity.

i. Transposon Tagging

In one embodiment of the invention, transposon tagging is used to reduceor eliminate the dimerization domain activity of one or moredimerization domain polypeptide. Transposon tagging comprises insertinga transposon within an endogenous dimerization domain gene to reduce oreliminate expression of the dimerization domain polypeptide.“dimerization domain gene” is intended to mean the gene that encodes adimerization domain polypeptide according to the invention.

In this embodiment, the expression of one or more dimerization domainpolypeptide is reduced or eliminated by inserting a transposon within aregulatory region or coding region of the gene encoding the dimerizationdomain polypeptide. A transposon that is within an exon, intron, 5′ or3′ untranslated sequence, a promoter or any other regulatory sequence ofa dimerization domain gene may be used to reduce or eliminate theexpression and/or activity of the encoded dimerization domainpolypeptide.

Methods for the transposon tagging of specific genes in plants are wellknown in the art. See, for example, Maes, et al., (1999) Trends PlantSci. 4:90-96; Dharmapuri and Sonti, (1999) FEMS Microbiol. Lett.179:53-59; Meissner, et al., (2000) Plant J. 22:265-274; Phogat, et al.,(2000) J. Biosci. 25:57-63; Walbot, (2000) Curr. Opin. Plant Biol.2:103-107; Gai, et al., (2000) Nucleic Acids Res. 28:94-96; Fitzmaurice,et al., (1999) Genetics 153:1919-1928). In addition, the TUSC processfor selecting Mu insertions in selected genes has been described inBensen, et al., (1995) Plant Cell 7:75-84; Mena, et al., (1996) Science274:1537-1540 and U.S. Pat. No. 5,962,764, each of which is hereinincorporated by reference.

ii. Mutant Plants with Reduced Activity

Additional methods for decreasing or eliminating the expression ofendogenous genes in plants are also known in the art and can besimilarly applied to the instant invention. These methods include otherforms of mutagenesis, such as ethyl methanesulfonate-inducedmutagenesis, deletion mutagenesis and fast neutron deletion mutagenesisused in a reverse genetics sense (with PCR) to identify plant lines inwhich the endogenous gene has been deleted. For examples of thesemethods see, Ohshima, et al., (1998) Virology 243:472-481; Okubara, etal., (1994) Genetics 137:867-874 and Quesada, et al., (2000) Genetics154:421-436, each of which is herein incorporated by reference. Inaddition, a fast and automatable method for screening for chemicallyinduced mutations, TILLING (Targeting Induced Local Lesions In Genomes),using denaturing HPLC or selective endonuclease digestion of selectedPCR products is also applicable to the instant invention. See, McCallum,et al., (2000) Nat. Biotechnol. 18:455-457, herein incorporated byreference.

Mutations that impact gene expression or that interfere with thefunction (dimerization domain activity) of the encoded protein are wellknown in the art. Insertional mutations in gene exons usually result innull-mutants. Mutations in conserved residues are particularly effectivein inhibiting the dimerization domain activity of the encoded protein.Conserved residues of plant dimerization domain polypeptides suitablefor mutagenesis with the goal to eliminate dimerization domain activityhave been described. Such mutants can be isolated according towell-known procedures and mutations in different dimerization domainloci can be stacked by genetic crossing. See, for example, Gruis, etal., (2002) Plant Cell 14:2863-2882.

In another embodiment of this invention, dominant mutants can be used totrigger RNA silencing due to gene inversion and recombination of aduplicated gene locus. See, for example, Kusaba, et al., (2003) PlantCell 15:1455-1467.

The invention encompasses additional methods for reducing or eliminatingthe activity of one or more dimerization domain polypeptide. Examples ofother methods for altering or mutating a genomic nucleotide sequence ina plant are known in the art and include, but are not limited to, theuse of RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repairvectors, mixed-duplex oligonucleotides, self-complementary RNA:DNAoligonucleotides and recombinogenic oligonucleobases. Such vectors andmethods of use are known in the art. See, for example, U.S. Pat. Nos.5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972 and 5,871,984,each of which are herein incorporated by reference. See also, WO98/49350, WO 99/07865, WO 99/25821 and Beetham, et al., (1999) Proc.Natl. Acad. Sci. USA 96:8774-8778, each of which is herein incorporatedby reference.

iii. Modulating Plant Growth and/or Dimerization Domain Component StackActivity

In specific methods, the level and/or activity of a dimerization domaingene in a plant is increased by increasing the level or activity of thedimerization domain polypeptide in the plant. Methods for increasing thelevel and/or activity of dimerization domain polypeptides in a plant arediscussed elsewhere herein. Briefly, such methods comprise providing adimerization domain polypeptide of the invention to a plant and therebyincreasing the level and/or activity of the dimerization domainpolypeptide. In other embodiments, a dimerization domain nucleotidesequence encoding a dimerization domain polypeptide can be provided byintroducing into the plant a polynucleotide comprising a dimerizationdomain nucleotide sequence of the invention, expressing the dimerizationdomain sequence, increasing the activity of the dimerization domainpolypeptide and thereby increasing the dimerization domain activity andtherefore the tissue growth in the plant or plant part. In otherembodiments, the dimerization domain nucleotide construct introducedinto the plant is stably incorporated into the genome of the plant.

In other methods, the number of cells and biomass of a plant tissue isincreased by increasing the level and/or activity of the dimerizationdomain polypeptide in the plant. Such methods are disclosed in detailelsewhere herein. In one such method, a dimerization domain nucleotidesequence is introduced into the plant and expression of saiddimerization domain nucleotide sequence decreases the activity of thedimerization domain polypeptide and thereby increasing the plant growthand/or dimerization domain in the plant or plant part. In otherembodiments, the dimerization domain nucleotide construct introducedinto the plant is stably incorporated into the genome of the plant.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate the level/activity of a plant growth and/ordimerization domain polynucleotide and polypeptide in the plant.Exemplary promoters for this embodiment have been disclosed elsewhereherein.

Accordingly, the present invention further provides plants having amodified plant growth and/or dimerization domain when compared to theplant growth and/or dimerization domain of a control plant tissue. Inone embodiment, the plant of the invention has an increasedlevel/activity of the dimerization domain polypeptide of the inventionand thus has increased plant growth and/or dimerization domain in theplant tissue. In other embodiments, the plant of the invention has areduced or eliminated level of the dimerization domain polypeptide ofthe invention and thus has decreased plant growth and/or dimerizationdomain in the plant tissue. In other embodiments, such plants havestably incorporated into their genome a nucleic acid molecule comprisinga dimerization domain nucleotide sequence of the invention operablylinked to a promoter that drives expression in the plant cell.

iv. Modulating Root Development

Methods for modulating root development in a plant are provided. By“modulating root development” is intended any alteration in thedevelopment of the plant root when compared to a control plant. Suchalterations in root development include, but are not limited to,alterations in the growth rate of the primary root, the fresh rootweight, the extent of lateral and adventitious root formation, thevasculature system, meristem development or radial expansion. Inparticular, the most desirable outcome would be a root with a strongervasculature that improves the standability of the plant and thus reducesroot lodging as well as being less susceptible to pests.

Methods for modulating root development in a plant are provided. Themethods comprise modulating the level and/or activity of thedimerization domain polypeptide in the plant. In one method, adimerization domain sequence of the invention is provided to the plant.In another method, the dimerization domain nucleotide sequence isprovided by introducing into the plant a polynucleotide comprising adimerization domain nucleotide sequence of the invention, expressing thedimerization domain sequence and thereby modifying root development. Instill other methods, the dimerization domain nucleotide constructintroduced into the plant is stably incorporated into the genome of theplant.

In other methods, root development is modulated by altering the level oractivity of the dimerization domain polypeptide in the plant. Anincrease in dimerization domain activity can result in at least one ormore of the following alterations to root development, including, butnot limited to, larger root meristems, increased in root growth,enhanced radial expansion, an enhanced vasculature system, increasedroot branching, more adventitious roots and/or an increase in fresh rootweight when compared to a control plant.

As used herein, “root growth” encompasses all aspects of growth of thedifferent parts that make up the root system at different stages of itsdevelopment in both monocotyledonous and dicotyledonous plants. It is tobe understood that enhanced root growth can result from enhanced growthof one or more of its parts including the primary root, lateral roots,adventitious roots, etc.

Methods of measuring such developmental alterations in the root systemare known in the art. See, for example, US Patent Application Number2003/0074698 and Werner, et al., (2001) PNAS 18:10487-10492, both ofwhich are herein incorporated by reference.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate root development in the plant. Exemplary promotersfor this embodiment include constitutive promoters and root-preferredpromoters. Exemplary root-preferred promoters have been disclosedelsewhere herein.

Stimulating root growth and increasing root mass by increasing theactivity and/or level of the dimerization domain polypeptide also findsuse in improving the standability of a plant. The term “resistance tolodging” or “standability” refers to the ability of a plant to fixitself to the soil. For plants with an erect or semi-erect growth habit,this term also refers to the ability to maintain an upright positionunder adverse (environmental) conditions. This trait relates to thesize, depth and morphology of the root system. In addition, stimulatingroot growth and increasing root mass by increasing the level and/oractivity of the dimerization domain polypeptide also finds use inpromoting in vitro propagation of explants.

Furthermore, higher root biomass production due to an increased leveland/or activity of dimerization domain activity has a direct effect onthe yield and an indirect effect of production of compounds produced byroot cells or transgenic root cells or cell cultures of said transgenicroot cells. One example of an interesting compound produced in rootcultures is shikonin, the yield of which can be advantageously enhancedby said methods.

Accordingly, the present invention further provides plants havingmodulated root development when compared to the root development of acontrol plant. In some embodiments, the plant of the invention has anincreased level/activity of the dimerization domain polypeptide of theinvention and has enhanced root growth and/or root biomass. In otherembodiments, such plants have stably incorporated into their genome anucleic acid molecule comprising a dimerization domain nucleotidesequence of the invention operably linked to a promoter that drivesexpression in the plant cell.

v. Modulating Shoot and Leaf Development

Methods are also provided for modulating shoot and leaf development in aplant. By “modulating shoot and/or leaf development” is intended anyalteration in the development of the plant shoot and/or leaf. Suchalterations in shoot and/or leaf development include, but are notlimited to, alterations in shoot meristem development, in leaf number,leaf size, leaf and stem vasculature, internode length and leafsenescence. As used herein, “leaf development” and “shoot development”encompasses all aspects of growth of the different parts that make upthe leaf system and the shoot system, respectively, at different stagesof their development, both in monocotyledonous and dicotyledonousplants. Methods for measuring such developmental alterations in theshoot and leaf system are known in the art. See, for example, Werner, etal., (2001) PNAS 98:10487-10492 and US Patent Application PublicationNumber 2003/0074698, each of which is herein incorporated by reference.

The method for modulating shoot and/or leaf development in a plantcomprises modulating the activity and/or level of a dimerization domainpolypeptide of the invention. In one embodiment, a dimerization domainsequence of the invention is provided. In other embodiments, thedimerization domain nucleotide sequence can be provided by introducinginto the plant a polynucleotide comprising a dimerization domainnucleotide sequence of the invention, expressing the dimerization domainsequence and thereby modifying shoot and/or leaf development. In otherembodiments, the dimerization domain nucleotide construct introducedinto the plant is stably incorporated into the genome of the plant.

In specific embodiments, shoot or leaf development is modulated bydecreasing the level and/or activity of the dimerization domainpolypeptide in the plant. An decrease in dimerization domain activitycan result in at least one or more of the following alterations in shootand/or leaf development, including, but not limited to, reduced leafnumber, reduced leaf surface, reduced vascular, shorter internodes andstunted growth and retarded leaf senescence when compared to a controlplant.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate shoot and leaf development of the plant. Exemplarypromoters for this embodiment include constitutive promoters,shoot-preferred promoters, shoot meristem-preferred promoters andleaf-preferred promoters. Exemplary promoters have been disclosedelsewhere herein.

Decreasing dimerization domain activity and/or level in a plant resultsin shorter internodes and stunted growth. Thus, the methods of theinvention find use in producing dwarf plants. In addition, as discussedabove, modulation of dimerization domain activity in the plant modulatesboth root and shoot growth. Thus, the present invention further providesmethods for altering the root/shoot ratio. Shoot or leaf development canfurther be modulated by decreasing the level and/or activity of thedimerization domain polypeptide in the plant.

Accordingly, the present invention further provides plants havingmodulated shoot and/or leaf development when compared to a controlplant. In some embodiments, the plant of the invention has an increasedlevel/activity of the dimerization domain polypeptide of the invention,altering the shoot and/or leaf development. Such alterations include,but are not limited to, increased leaf number, increased leaf surface,increased vascularity, longer internodes and increased plant stature, aswell as alterations in leaf senescence, as compared to a control plant.In other embodiments, the plant of the invention has a decreasedlevel/activity of the dimerization domain polypeptide of the invention.

vi Modulating Reproductive Tissue Development

Methods for modulating reproductive tissue development are provided. Inone embodiment, methods are provided to modulate floral development in aplant. By “modulating floral development” is intended any alteration ina structure of a plant's reproductive tissue as compared to a controlplant in which the activity or level of the dimerization domainpolypeptide has not been modulated. “Modulating floral development”further includes any alteration in the timing of the development of aplant's reproductive tissue (i.e., a delayed or an accelerated timing offloral development) when compared to a control plant in which theactivity or level of the dimerization domain polypeptide has not beenmodulated. Macroscopic alterations may include changes in size, shape,number or location of reproductive tissues, the developmental timeperiod that these structures form or the ability to maintain or proceedthrough the flowering process in times of environmental stress.Microscopic alterations may include changes to the types or shapes ofcells that make up the reproductive tissues.

The method for modulating floral development in a plant comprisesmodulating dimerization domain activity in a plant. In one method, adimerization domain sequence of the invention is provided. Adimerization domain nucleotide sequence can be provided by introducinginto the plant a polynucleotide comprising a dimerization domainnucleotide sequence of the invention, expressing the dimerization domainsequence and thereby modifying floral development. In other embodiments,the dimerization domain nucleotide construct introduced into the plantis stably incorporated into the genome of the plant.

In specific methods, floral development is modulated by decreasing thelevel or activity of the dimerization domain polypeptide in the plant. Adecrease in dimerization domain activity can result in at least one ormore of the following alterations in floral development, including, butnot limited to, retarded flowering, reduced number of flowers, partialmale sterility and reduced seed set when compared to a control plant.Inducing delayed flowering or inhibiting flowering can be used toenhance yield in forage crops such as alfalfa. Methods for measuringsuch developmental alterations in floral development are known in theart. See, for example, Mouradov, et al., (2002) The Plant CellS111-S130, herein incorporated by reference.

As discussed above, one of skill will recognize the appropriate promoterto use to modulate floral development of the plant. Exemplary promotersfor this embodiment include constitutive promoters, inducible promoters,shoot-preferred promoters and inflorescence-preferred promoters.

In other methods, floral development is modulated by increasing thelevel and/or activity of the dimerization domain sequence of theinvention. Such methods can comprise introducing a dimerization domainnucleotide sequence into the plant and increasing the activity of thedimerization domain polypeptide. In other methods, the dimerizationdomain nucleotide construct introduced into the plant is stablyincorporated into the genome of the plant. Increasing expression of thedimerization domain sequence of the invention can modulate floraldevelopment during periods of stress. Such methods are describedelsewhere herein. Accordingly, the present invention further providesplants having modulated floral development when compared to the floraldevelopment of a control plant. Compositions include plants having anincreased level/activity of the dimerization domain polypeptide of theinvention and having an altered floral development. Compositions alsoinclude plants having an increased level/activity of the dimerizationdomain polypeptide of the invention wherein the plant maintains orproceeds through the flowering process in times of stress.

Methods are also provided for the use of the dimerization domain of theinvention to increase seed size and/or weight. The method comprisesincreasing the activity of the dimerization domain in a plant or plantpart, such as the seed. An increase in seed size and/or weight comprisesan increased size or weight of the seed and/or an increase in the sizeor weight of one or more seed part including, for example, the embryo,endosperm, seed coat, aleurone or cotyledon.

As discussed above, one of skill will recognize the appropriate promoterto use to increase seed size and/or seed weight. Exemplary promoters ofthis embodiment include constitutive promoters, inducible promoters,seed-preferred promoters, embryo-preferred promoters andendosperm-preferred promoters.

The method for decreasing seed size and/or seed weight in a plantcomprises decreasing dimerization domain activity in the plant. In oneembodiment, the dimerization domain nucleotide sequence can be providedby introducing into the plant a polynucleotide comprising a dimerizationdomain nucleotide sequence of the invention, expressing the dimerizationdomain sequence and thereby increasing seed weight and/or size. In otherembodiments, the dimerization domain nucleotide construct introducedinto the plant is stably incorporated into the genome of the plant.

It is further recognized that increasing seed size and/or weight canalso be accompanied by an increase in the speed of growth of seedlingsor an increase in early vigor. As used herein, the term “early vigor”refers to the ability of a plant to grow rapidly during earlydevelopment and relates to the successful establishment, aftergermination, of a well-developed root system and a well-developedphotosynthetic apparatus. In addition, an increase in seed size and/orweight can also result in an increase in plant yield when compared to acontrol.

Accordingly, the present invention further provides plants having anincreased seed weight and/or seed size when compared to a control plant.In other embodiments, plants having an increased vigor and plant yieldare also provided. In some embodiments, the plant of the invention hasan increased level/activity of the dimerization domain polypeptide ofthe invention and has an increased seed weight and/or seed size. Inother embodiments, such plants have stably incorporated into theirgenome a nucleic acid molecule comprising a dimerization domainnucleotide sequence of the invention operably linked to a promoter thatdrives expression in the plant cell.

vii. Method of Use for Dimerization Domain Promoter Polynucleotides

The polynucleotides comprising the dimerization domain promotersdisclosed in the present invention, as well as variants and fragmentsthereof, are useful in the genetic manipulation of any host cell,preferably plant cell, when assembled with a DNA construct such that thepromoter sequence is operably linked to a nucleotide sequence comprisinga polynucleotide of interest. In this manner, the dimerization domainpromoter polynucleotides of the invention are provided in expressioncassettes along with a polynucleotide sequence of interest forexpression in the host cell of interest. The dimerization domainpromoter sequences of the invention are expressed in a variety oftissues containing cells that have dimerization domain and thus thepromoter sequences can find use in regulating the temporal and/or thespatial expression of polynucleotides of interest particularly in thedimerization domain containing cells.

Synthetic hybrid promoter regions are known in the art. Such regionscomprise upstream promoter elements of one polynucleotide operablylinked to the promoter element of another polynucleotide. In anembodiment of the invention, heterologous sequence expression iscontrolled by a synthetic hybrid promoter comprising the dimerizationdomain promoter sequences of the invention or a variant or fragmentthereof, operably linked to upstream promoter element(s) from aheterologous promoter. Upstream promoter elements that are involved inthe plant defense system have been identified and may be used togenerate a synthetic promoter. See, for example, Rushton, et al., (1998)Curr. Opin. Plant Biol. 1:311-315. Alternatively, a syntheticdimerization domain promoter sequence may comprise duplications of theupstream promoter elements found within the dimerization domain promotersequences.

It is recognized that the promoter sequence of the invention may be usedwith its native dimerization domain coding sequences. A DNA constructcomprising the dimerization domain promoter operably linked with itsnative dimerization domain gene may be used to transform any plant ofinterest to bring about a desired phenotypic change, such as modulatingcell number, modulating root, shoot, leaf, floral and embryodevelopment, stress tolerance and any other phenotype describedelsewhere herein.

The promoter nucleotide sequences and methods disclosed herein areuseful in regulating expression of any heterologous nucleotide sequencein a host plant in order to vary the phenotype of a plant. Variouschanges in phenotype are of interest including modifying the fatty acidcomposition in a plant, altering the amino acid content of a plant,altering a plant's pathogen defense mechanism, and the like. Theseresults can be achieved by providing expression of heterologous productsor increased expression of endogenous products in plants. Alternatively,the results can be achieved by providing for a reduction of expressionof one or more endogenous products, particularly enzymes or cofactors inthe plant. These changes result in a change in phenotype of thetransformed plant.

Genes of interest are reflective of the commercial markets and interestsof those involved in the development of the crop. Crops and markets ofinterest change, and as developing nations open up world markets, newcrops and technologies will emerge also. In addition, as ourunderstanding of agronomic traits and characteristics such as yield andheterosis increase, the choice of genes for transformation will changeaccordingly. General categories of genes of interest include, forexample, those genes involved in information, such as zinc fingers,those involved in communication, such as kinases and those involved inhousekeeping, such as heat shock proteins. More specific categories oftransgenes, for example, include genes encoding important traits foragronomics, insect resistance, disease resistance, herbicide resistance,sterility, grain characteristics and commercial products. Genes ofinterest include, generally, those involved in oil, starch, carbohydrateor nutrient metabolism as well as those affecting kernel size, sucroseloading and the like.

In certain embodiments the nucleic acid sequences of the presentinvention can be used in combination (“stacked”) with otherpolynucleotide sequences of interest in order to create plants with adesired phenotype. The combinations generated can include multiplecopies of any one or more of the polynucleotides of interest. Thepolynucleotides of the present invention may be stacked with any gene orcombination of genes to produce plants with a variety of desired traitcombinations, including but not limited to traits desirable for animalfeed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balancedamino acids (e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801;5,885,802 and 5,703,409); barley high lysine (Williamson, et al., (1987)Eur. J. Biochem. 165:99-106 and WO 98/20122) and high methionineproteins (Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, etal., (1988) Gene 71:359 and Musumura, et al., (1989) Plant Mol. Biol.12:123)); increased digestibility (e.g., modified storage proteins (U.S.patent application Ser. No. 10/053,410, filed Nov. 7, 2001) andthioredoxins (U.S. patent application Ser. No. 10/005,429, filed Dec. 3,2001)), the disclosures of which are herein incorporated by reference.The polynucleotides of the present invention can also be stacked withtraits desirable for insect, disease or herbicide resistance (e.g.,Bacillus thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892;5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser, et al., (1986) Gene48:109); lectins (Van Damme, et al., (1994) Plant Mol. Biol. 24:825);fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence anddisease resistance genes (Jones, et al., (1994) Science 266:789; Martin,et al., (1993) Science 262:1432; Mindrinos, et al., (1994) Cell78:1089); acetolactate synthase (ALS) mutants that lead to herbicideresistance such as the S4 and/or Hra mutations; inhibitors of glutaminesynthase such as phosphinothricin or basta (e.g., bar gene); andglyphosate resistance (EPSPS gene)) and traits desirable for processingor process products such as high oil (e.g., U.S. Pat. No. 6,232,529);modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.5,952,544; WO 94/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE) and starch debranching enzymes (SDBE)) and polymers orbioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,polyhydroxybutyrate synthase and acetoacetyl-CoA reductase (Schubert, etal., (1988) J. Bacteriol. 170:5837-5847) facilitate expression ofpolyhydroxyalkanoates (PHAs)), the disclosures of which are hereinincorporated by reference. One could also combine the polynucleotides ofthe present invention with polynucleotides affecting agronomic traitssuch as male sterility (e.g., see, U.S. Pat. No. 5,583,210), stalkstrength, flowering time or transformation technology traits such ascell cycle regulation or gene targeting (e.g., WO 99/61619; WO 00/17364;WO 99/25821), the disclosures of which are herein incorporated byreference.

In one embodiment, sequences of interest improve plant growth and/orcrop yields. For example, sequences of interest include agronomicallyimportant genes that result in improved primary or lateral root systems.Such genes include, but are not limited to, nutrient/water transportersand growth induces. Examples of such genes, include but are not limitedto, maize plasma membrane H⁺-ATPase (MHA2) (Frias, et al., (1996) PlantCell 8:1533-44); AKT1, a component of the potassium uptake apparatus inArabidopsis, (Spalding, et al., (1999) J Gen Physiol 113:909-18); RMLgenes which activate cell division cycle in the root apical cells(Cheng, et al., (1995) Plant Physiol 108:881); maize glutaminesynthetase genes (Sukanya, et al., (1994) Plant Mol Biol 26:1935-46) andhemoglobin (Duff, et al., (1997) J. Biol. Chem 27:16749-16752,Arredondo-Peter, et al., (1997) Plant Physiol. 115:1259-1266;Arredondo-Peter, et al., (1997) Plant Physiol 114:493-500 and referencessited therein). The sequence of interest may also be useful inexpressing antisense nucleotide sequences of genes that that negativelyaffects root development.

Additional, agronomically important traits such as oil, starch andprotein content can be genetically altered in addition to usingtraditional breeding methods. Modifications include increasing contentof oleic acid, saturated and unsaturated oils, increasing levels oflysine and sulfur, providing essential amino acids and also modificationof starch. Hordothionin protein modifications are described in U.S. Pat.Nos. 5,703,049, 5,885,801, 5,885,802 and 5,990,389, herein incorporatedby reference. Another example is lysine and/or sulfur rich seed proteinencoded by the soybean 2S albumin described in U.S. Pat. No. 5,850,016and the chymotrypsin inhibitor from barley, described in Williamson, etal., (1987) Eur. J. Biochem. 165:99-106, the disclosures of which areherein incorporated by reference.

Derivatives of the coding sequences can be made by site-directedmutagenesis to increase the level of preselected amino acids in theencoded polypeptide. For example, the gene encoding the barley highlysine polypeptide (BHL) is derived from barley chymotrypsin inhibitor,U.S. patent application Ser. No. 08/740,682, filed Nov. 1, 1996 and WO98/20133, the disclosures of which are herein incorporated by reference.Other proteins include methionine-rich plant proteins such as fromsunflower seed (Lilley, et al., (1989) Proceedings of the World Congresson Vegetable Protein Utilization in Human Foods and Animal Feedstuffs,ed. Applewhite (American Oil Chemists Society, Champaign, Ill.), pp.497-502; herein incorporated by reference); corn (Pedersen, et al.,(1986) J. Biol. Chem. 261:6279; Kirihara, et al., (1988) Gene 71:359,both of which are herein incorporated by reference) and rice (Musumura,et al., (1989) Plant Mol. Biol. 12:123, herein incorporated byreference). Other agronomically important genes encode latex, Floury 2,growth factors, seed storage factors and transcription factors.

Insect resistance genes may encode resistance to pests that have greatyield drag such as rootworm, cutworm, European Corn Borer and the like.Such genes include, for example, Bacillus thuringiensis toxic proteingenes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756;5,593,881 and Geiser, et al., (1986) Gene 48:109), and the like.

Genes encoding disease resistance traits include detoxification genes,such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr)and disease resistance (R) genes (Jones, et al., (1994) Science 266:789;Martin, et al., (1993) Science 262:1432 and Mindrinos, et al., (1994)Cell 78:1089), and the like.

Herbicide resistance traits may include genes coding for resistance toherbicides that act to inhibit the action of acetolactate synthase(ALS), in particular the sulfonylurea-type herbicides (e.g., theacetolactate synthase (ALS) gene containing mutations leading to suchresistance, in particular the S4 and/or Hra mutations), genes coding forresistance to herbicides that inhibit enol-pyruvylshikimate phosphatesynthase (EPSPS), e.g., glyphosate acetyl transferase (GAT), genescoding for resistance to herbicides that act to inhibit action ofglutamine synthase, such as phosphinothricin or basta (e.g., the bargene), a combination thereof or other such genes known in the art. Thebar gene encodes resistance to the herbicide basta, the nptII geneencodes resistance to the antibiotics kanamycin and geneticin and theALS-gene mutants encode resistance to the herbicide chlorsulfuron.

Sterility genes can also be encoded in an expression cassette andprovide an alternative to physical detasseling. Examples of genes usedin such ways include male tissue-preferred genes and genes with malesterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210.Other genes include kinases and those encoding compounds toxic to eithermale or female gametophytic development.

The quality of grain is reflected in traits such as levels and types ofoils, saturated and unsaturated, quality and quantity of essential aminoacids and levels of cellulose. In corn, modified hordothionin proteinsare described in U.S. Pat. Nos. 5,703,049, 5,885,801, 5,885,802 and5,990,389.

Commercial traits can also be encoded on a gene or genes that couldincrease for example, starch for ethanol production, or provideexpression of proteins. Another important commercial use of transformedplants is the production of polymers and bioplastics such as describedin U.S. Pat. No. 5,602,321. Genes such as 13-Ketothiolase, PHBase(polyhydroxyburyrate synthase) and acetoacetyl-CoA reductase (see,Schubert, et al., (1988) J. Bacteriol. 170:5837-5847) facilitateexpression of polyhyroxyalkanoates (PHAs).

Exogenous products include plant enzymes and products as well as thosefrom other sources including procaryotes and other eukaryotes. Suchproducts include enzymes, cofactors, hormones and the like. The level ofproteins, particularly modified proteins having improved amino aciddistribution to improve the nutrient value of the plant, can beincreased. This is achieved by the expression of such proteins havingenhanced amino acid content.

This invention can be better understood by reference to the followingnon-limiting examples. It will be appreciated by those skilled in theart that other embodiments of the invention may be practiced withoutdeparting from the spirit and the scope of the invention as hereindisclosed and claimed.

Examples Example 1 Yield and Harvest Index Tests—RT810ZBS_T(Intro-EF09B/GR1B5)

-   -   PHP26963 (S2a:D8MPL+S2a:CesA10)—10 evts    -   PHP26998 (S2a:D8mpl+Nas2:DD+S2a:CesA10)—8 evts    -   PHP24843 (S2a:D8MPL+NAS2:DD)—4 evts    -   Construct Nulls (3 events/null)    -   WT (Intro-EF09BZTZ/GR1B5) 2 Densities    -   36,000 PPA (JH, MR)—Yield (5 reps)    -   48,000 PPA (JH, MR)—Yield (5 reps), Harvest Index (3 reps)

Three constructs were tested at Johnston (JH) and Marion (MR) Iowa attwo densities, 36,000 plants per acre (PPA) and 48,000 PPA in 20″ rowwidth. The genes tested consisted of the dwarf mutant D8mpl and theadditional genes (stacks) DD (dimerization domain of the D8 gene) or theCes A10 gene. The constructs are shown below in which the transgenicevents are shown with their plasmid identification and nulls(segregating non transgenic sibs) are shown with their plasmiddesignation and the letter n.

TABLE 2 D8mpl + DD php24843 E7216.51.1.1 php24843n E7216.49.1.5 D8 +CesA php26963 E7216.49.2.1 php26963 E7216.49.2.2 php26963 E7216.49.3.1php26963n CN D8 + DD + CesA php26998 E7216.50.1.1 php26998 E7216.50.1.3php26998n CN

A higher plant population density was chosen to determine if the dwarfand dwarf stack transgenic plants behaved the same or differently thanthe construct null sibs (no transgenes with normal height) for yield andharvest index. In general, corn shows a decline in yield in populationsabove the optimum economic yield such that yield levels decline. Harvestindex in corn has been relatively stable, from 45 to 50% as defined byear dry matter/total above ground dry matter. Increases in biomass andharvest index are the major determinants of yield, thus a positivechange in either attribute could lead to higher potential yield.

The yield comparison for selected events of the different constructs atthe Johnston and Marion locations was performed. In general, thereduction in yield levels due to high plant population was morepronounced in the null sibs compared to the respective transgenic stackconstructs. In some instances, the transgenic treatments showed anunexpected and increased yield response, particularly in the Marion,Iowa location. Such an observation indicates that further breeding witha variety of different germplasm sources in addition to those used withthese transgene stacks or additional optimization of the agronomicfactors such as row width, fertilization practices or optimized plantpopulation for the dwarf phenotype would further improve yieldpotential.

Harvest index of the entries at the higher population density of 48,000PPA was measured. Generally, the harvest index of the null sibs werejust between 0.5 and 0.52 while most of the transgenic stacks had aharvest index in the range of 0.54 to 0.58. The increase in harvestindex could be expected to make better use of available soil moistureand nutrients since a greater proportion of the dry matter produced isin the form of grain.

Example 2 Yield and Harvest Index Tests—(Intro-EF09B/HG11)

Topcrosses were made from PHP26963, PHP26998 and PHP24843 T0 plants ontoHG11 females. This produced a background genotype similar to commercialhybrid 33A14 which could be used as a reference. These plants were thengrown in Johnston observation plots. A small planting of PHP17881hybrids were also included.

-   -   PHP26963 (S2a:D8MPL+S2a:CesA10)—2 evts, 6 rows    -   PHP26998 (S2a:D8mpl+Nas2:DD+S2a:CesA10)—2 evts, 6 rows    -   PHP24843 (S2a:D8MPL+NAS2:DD)—2 evts, 8 rows    -   PHP17881 (S2a:D8MPL)    -   WT (EF09B/HG11-33A14)—11 rows

Minirhizotron tubes were inserted in the soil near these plants to allowfor imaging of roots that intersected the tubes. This allowed for adirect measurement of root length of the NAS2:DD stack constructs (FIGS.2-5). The DD stacks had longer root systems at earlier time points andappeared to be colonizing the soil more rapidly than the non DDcounterparts. The later time point showed the non-DD constructs havingsimilar root lengths to the DD stacks at more shallow depths, but notyet fully colonizing the lowest depth of soil measured. The surface areaof these plants increased proportionately with the length, indicatingthat there is no sacrifice of root width. Plant height and yields wereconsistent with previous observations of S2a:D8MPL constructs (FIG. 4and Table 3—Heights from field experiments).

TABLE 3 Average Height Standard Deviation (m) (m) 33A14 2.90 0.06PHP17881 (D8 MPL) 1.99 0.28 PHP24843 (D8/DD) 1.96 0.06 PHP26963 (D8/CES)2.10 0.09 PHP26998(D8/DD/CES) 1.90 0.10

Example 3 Greenhouse Grown Transgenic Stacks

Three constructs were tested in the introEF09B background at the T0generation to determine the agronomic characteristics of the D8dimerization domain stacks and for preparation for field testing. Eachstacked construct (PHP24843, PHP26963 and PHP26998) utilized the S2APRO:D8MPL gene, NAS2 PRO:D8 243-331 and/or S2A PRO:ZM-CES A10.

-   -   Genes Tested (Intro EF09B)        -   S2a:D8MPL (Vascular Element Preferred Promoter:moderate            dwarfing Gene)        -   Nas2:DD (Root Preferred Promoter: Leucine Zipper            Dimerization Domain)        -   S2a:CesA10 (Vascular Element Preferred Promoter:Cellulose            Synthase Gene in Stalk Tissue.    -   Gene Combinations (“Stacks”) of Two and Three Genes        -   PHP24843—NAS2 PRO:D8 243-331/S2A PRO:D8MPL Stack (13 events)        -   PHP26963—S2A PRO:ZM-CES A10/S2A PRO:D8MPL Stack (15 events)        -   PHP26998—NAS2 PRO:D8 243-331/S2A PRO:ZM-CES A10/S2A            PRO:D8MPL (14 events)

Morphometric analyses were performed on the mature T0 plants from thisexperiment (FIG. 6). The NAS2 PRO:D8 243-331 gene increased leaf widthand area in this experiment. The S2A PRO:ZM-CES A10 gene increased leafangle, decreased leaf length and increased seed number.

Example 4 Greenhouse Grown Transgenic Stacks

Five constructs were tested in GS3×GF3 at the T0 generation to determinethe effectiveness of the D8 dimerization domain for reversing dwarfingof the maize root system. Each stacked construct (PHP24843, PHP24844 andPHP24861) utilized a different root preferred promoter to driveexpression the D8 243-331 coding sequence.

-   -   Genes Tested (GS3×GF#)        -   S2a:D8MPL (Vascular Element Preferred Promoter:moderate            dwarfing Gene)        -   Nas2:DD (Root Preferred Promoter: Leucine Zipper            Dimerization Domain)        -   ZRP2.47 PRO:D8 243-331 (Root Preferred Promoter: Leucine            Zipper Dimerization Domain)        -   ROOTMET2 PRO:D8 243-331 (Root Preferred Promoter: Leucine            Zipper Dimerization Domain)        -   ROOTMET2 PRO:GUSINT (Root Preferred Promoter:            β-glucuronidase reporter gene)    -   Gene Combinations (“Stacks”) of Two Genes        -   PHP24843—NAS2 PRO:D8 243-331/S2A PRO:D8MPL Stack (25 events)        -   PHP24844—ZRP2.47 PRO:D8 243-331/S2A PRO:D8MPL Stack (23            events)        -   PHP24861—ROOTMET2 PRO:D8 243-331/S2A PRO:D8MPL Stack (22            events)        -   PHP17881—S2A PRO:D8MPL (Dwarf Control) (14 events)        -   PHP23206—ROOTMET2 PRO:GUSINT (Full Size Control) (14 events)

Morphometric analyses were performed on the mature T0 plants from thisexperiment (FIG. 7). The findings were that root weight was notsignificantly altered. The expected root change is in root length, whichcould not be measured due to root bound growth in greenhouse pots. Eachconstruct with the S2A PRO:D8MPL gene displayed a reduced stature withplant height reduced by ˜25-35%. Stalk weight was lower in thedimerization domain constructs than in the S2A PRO:D8MPL alone, whichwas in-turn lower than the full size control. Leaf weight and seednumber were reduced in PHP24844, PHP24861 and PHP17881 compared to thefull size control; however, PHP24843 (NAS2 PRO:D8 243-331/S2A PRO:D8MPLStack) retained leaf weight and seed numbers equal to those of the fullsize control. Seed number is a component of yield and stalk weight is acomponent of biomass, indicating that PHP24843 may increase harvestindex.

Example 5 Greenhouse Grown D8 Dimerization Domain Transgenics

Four constructs were tested in GS3×GF3 at the T0 generation to determinethe effects of the D8 dimerization domain when expressed in roots in anon-stacked configuration. Each stacked construct (PHP24711, PHP24712and PHP24713) utilized a different root preferred promoter to driveexpression the D8 243-331 coding sequence.

-   -   Genes Tested (GS3×GF#)        -   Nas2:DD (Root Preferred Promoter: Leucine Zipper            Dimerization Domain)        -   ZRP2.47 PRO:D8 243-331 (Root Preferred Promoter: Leucine            Zipper Dimerization Domain)        -   ROOTMET2 PRO:D8 243-331 (Root Preferred Promoter: Leucine            Zipper Dimerization Domain)    -   Gene Constructs        -   PHP24711—ZRP2.47 PRO:D8 243-331 (25 events)        -   PHP24712—ROOTMET2 PRO:D8 243-331 (25 events)        -   PHP24713—NAS2 PRO:D8 243-331 (25 events)        -   PHP24715—S2A PRO:AC-GFP1 (Full Size Control) (25 events)

Morphometric analyses were performed on the mature T0 plants from thisexperiment (FIG. 8). Stalk weight and seed number were increased in theROOTMET2 PRO:D8 243-331 and NAS2 PRO:D8 243-331 constructs.

Example 6 Two Location, 3 Construct, Yield and Harvest Index Trial

Yield and harvest index comparisons were made with three differentconstructs in genotype “Intro EF09B/GR1B5” compared to their respectiveconstruct nulls. The data is described in Table 4. The yield and harvestindex was measured in replicated experiments (5) at 48,000 PPA seeded in20″ rows in Johnston and Marion Iowa using a randomized complete blockdesign. Generally, the semi-dwarf plant height was approximately 60-70%of the construct nulls (CN) with each construct. The phP29693 andphP26998 plant height was about 65% (about 12″ taller) than phP24843.Compared to the construct nulls, yield in Johnston was near equal to theconstruct null with the events shown in this table. Harvest index wassignificantly higher in the Johnston location. At the Marion location,several constructs/events were higher in yield than their respectiveconstruct nulls. Harvest index was numerically higher and in most casessignificantly higher than their respective construct nulls. Thesemi-dwarf transgenics had a better yield response at high populationscompared to the construct nulls when grown at a lower population of36,000 ppa. The ‘stacked’ combinations of D8mpl+DD was not significantlylower than the construct null for yield but had higher harvest index atboth locations. The combination in construct phP26963 had higher yieldpotential in Johnston and Marion with higher harvest index. Thecombination of D8mpl+DD+CesA 10 had similar or equal yield and higherharvest index in Johnston while the triple gene stack in the Marion,Iowa location shows lower yields but higher harvest index. Althoughthere were some individual plots that showed root and stalk lodging,neither location had significant differences between the transgenic andtheir construct nulls.

TABLE 4 % JH % MR JH of HI MR of HI Gene PHP ID # yield null (%) yieldnull (%) 0.57 0.56 D8mpl/ control E7216.51.1.1 168  95% (110) 152  98%(103) DD CN 177   0.51 156 0.54   0.58 0.54 D8/ 26963 E7216.49.1.5 16392 (114) 141 105% (111) CesA   0.56 0.58 E7216.49.2.1 172  97% (111) 147110% (112) 0.56 0.53 E7216.49.2.2 176 100% (111) 152 113% (104) 0.530.55 E7216.49.3.1 176  99% (104) 151 113% (108) CN 177   0.51 134 0.51  0.56 0.58 D8/DD/ 26998 E7216.50.1.1 158  93% (109) 133  90% (113) CesA  0.57   0.54 E7216.50.1.3 168  99% (110) 133  90% (104) CN 169 0.51 1490.52 HI = Harvest Index JH = Johnston MR = Marion

Example 7 Transformation and Regeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing the Zm dimerization domain sequence operably linkedto the drought-inducible promoter RAB17 promoter (Vilardell, et al.,(1990) Plant Mol Biol 14:423-432) and the selectable marker gene PAT,which confers resistance to the herbicide Bialaphos. Alternatively, theselectable marker gene is provided on a separate plasmid. Transformationis performed as follows. Media recipes follow below.

Preparation of Target Tissue:

The ears are husked and surface sterilized in 30% Clorox® bleach plus0.5% Micro detergent for 20 minutes and rinsed two times with sterilewater. The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate, on 560Y medium for 4 hoursand then aligned within the 2.5-cm target zone in preparation forbombardment.

Preparation of DNA:

A plasmid vector comprising the dimerization domain sequence operablylinked to an ubiquitin promoter is made. This plasmid DNA plus plasmidDNA containing a PAT selectable marker is precipitated onto 1.1 μm(average diameter) tungsten pellets using a CaCl₂ precipitationprocedure as follows:

100 μl prepared tungsten particles in water

10 μl (1 μg) DNA in Tris EDTA buffer (1 μg total DNA)

100 μl 2.5M CaCl₂

10 μl 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol and centrifugedfor 30 seconds. Again the liquid is removed, and 105 μl 100% ethanol isadded to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment:

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment:

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos and subcultured every 2 weeks. After approximately 10 weeks ofselection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored and scored for increased drought tolerance. Assaysto measure improved drought tolerance are routine in the art andinclude, for example, increased kernel-earring capacity yields underdrought conditions when compared to control maize plants under identicalenvironmental conditions. Alternatively, the transformed plants can bemonitored for a modulation in meristem development (i.e., a decrease inspikelet formation on the ear). See, for example, Bruce, et al., (2002)Journal of Experimental Botany 53:1-13.

Bombardment and Culture Media:

Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts (SIGMAC-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000× SIGMA-1511), 0.5 mg/lthiamine HCl, 120.0 g/l sucrose, 1.0 mg/l 2,4-D and 2.88 g/l L-proline(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 2.0 g/l Gelrite® (added after bringing to volume with D-I H₂O) and8.5 mg/l silver nitrate (added after sterilizing the medium and coolingto room temperature). Selection medium (560R) comprises 4.0 g/l N6 basalsalts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix (1000×SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose and 2.0 mg/l 2,4-D(brought to volume with D-I H₂O following adjustment to pH 5.8 withKOH); 3.0 g/l Gelrite® (added after bringing to volume with D-I H₂O) and0.85 mg/l silver nitrate and 3.0 mg/l bialaphos (both added aftersterilizing the medium and cooling to room temperature).

Plant regeneration medium (288J) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g nicotinic acid,0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and 0.40 g/l glycinebrought to volume with polished D-I H₂O) (Murashige and Skoog, (1962)Physiol. Plant. 15:473), 100 mg/l myo-inositol, 0.5 mg/l zeatin, 60 g/lsucrose and 1.0 ml/l of 0.1 mM abscisic acid (brought to volume withpolished D-I H₂O after adjusting to pH 5.6); 3.0 g/l Gelrite® (addedafter bringing to volume with D-I H₂O) and 1.0 mg/l indoleacetic acidand 3.0 mg/l bialaphos (added after sterilizing the medium and coolingto 60° C.). Hormone-free medium (272V) comprises 4.3 g/l MS salts (GIBCO11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g/l nicotinicacid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and 0.40 g/lglycine brought to volume with polished D-I H₂O), 0.1 g/l myo-inositoland 40.0 g/l sucrose (brought to volume with polished D-I H₂O afteradjusting pH to 5.6) and 6 g/l Bacto™-agar (added after bringing tovolume with polished D-I H₂O), sterilized and cooled to 60° C.

Example 8 Agrobacterium-Mediated Transformation

For Agrobacterium-mediated transformation of maize with an antisensesequence of the Zmdimerization domain sequence of the present invention,preferably the method of Zhao is employed (U.S. Pat. No. 5,981,840 andPCT Patent Publication WO98/32326, the contents of which are herebyincorporated by reference). Briefly, immature embryos are isolated frommaize and the embryos contacted with a suspension of Agrobacterium,where the bacteria are capable of transferring the dimerization domainsequence to at least one cell of at least one of the immature embryos(step 1: the infection step). In this step the immature embryos arepreferably immersed in an Agrobacterium suspension for the initiation ofinoculation. The embryos are co-cultured for a time with theAgrobacterium (step 2: the co-cultivation step). Preferably the immatureembryos are cultured on solid medium following the infection step.Following this co-cultivation period an optional “resting” step iscontemplated. In this resting step, the embryos are incubated in thepresence of at least one antibiotic known to inhibit the growth ofAgrobacterium without the addition of a selective agent for planttransformants (step 3: resting step). Preferably the immature embryosare cultured on solid medium with antibiotic, but without a selectingagent, for elimination of Agrobacterium and for a resting phase for theinfected cells. Next, inoculated embryos are cultured on mediumcontaining a selective agent and growing transformed callus is recovered(step 4: the selection step). Preferably, the immature embryos arecultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus is then regeneratedinto plants (step 5: the regeneration step) and preferably calli grownon selective medium are cultured on solid medium to regenerate theplants. Plants are monitored and scored for a modulation in meristemdevelopment. For instance, alterations of size and appearance of theshoot and floral meristems and/or increased yields of leaves, flowersand/or fruits are monitored.

Example 9 Sugar Cane Transformation

This protocol describes routine conditions for production of transgenicsugarcane lines. The same conditions are close to optimal for number oftransiently expressing cells following bombardment into embryogenicsugarcane callus. See also, Bower, et al., (1996). Molec Breed2:239-249; Birch and Bower, (1994). Principles of gene transfer usingparticle bombardment. In Particle Bombardment Technology for GeneTransfer, Yang and Christou, eds (New York: Oxford University Press),pp. 3-37 and Santosa, et al., (2004), Molecular Biotechnology28:113-119, incorporated herein by reference.

Sugarcane Transformation Protocol: 1. Subculture Callus on MSC3, 4 DaysPrior to Bombardment:

-   -   (a) Use actively growing embryogenic callus (predominantly        globular pro-embryoids rather than more advanced stages of        differentiation) for bombardment and through the subsequent        selection period.    -   (b) Divide callus into pieces around 5 mm in diameter at the        time of subculture and use forceps to make a small crater in the        agar surface for each transferred callus piece.    -   (c) Incubate at 28° C. in the dark, in deep (25 mm) Petri dishes        with micropore tape seals for gas exchange.        2. Place embryogenic callus pieces in a circle (˜2.5 cm        diameter), on MSC3Osm medium. Incubate for 4 hours prior to        bombardment.        3. Sterilize 0.7 μm diameter tungsten (Grade M-10, Bio-Rad        #165-2266) in absolute ethanol. Vortex the suspension, then        pellet the tungsten in a microfuge for ˜30 seconds. Draw off the        supernatant and resuspend the particles at the same        concentration in sterile H₂0. Repeat the washing step with        sterile H₂0 twice and thoroughly resuspend particles before        transferring 50 μl aliquots into microfuge tubes.        4. Add the precipitation mix components:

Component (stock solution) Volume to add Final conc in mix Tungsten (100μg/μl in H₂0) 50 μl 38.5 μg/μl DNA (1 μg/μl) 10 μl 0.38 μg/μl CaCl₂(2.5M in H20) 50 μl 963 mM Spermidine free base (0.1M in H₂0) 20 μl 15mM5. Allow the mixture to stand on ice for 5 min. During this time,complete steps 6-8 below.6. Disinfect the inside of the ‘gene gun’ target chamber by swabbingwith ethanol and allow it to dry.7. Adjust the outlet pressure at the helium cylinder to the desiredbombardment pressure.8. Adjust the solenoid timer to 0.05 seconds. Pass enough helium toremove air from the supply line (2-3 pulses).9. After 5 min on ice, remove (and discard) 100 μl of supernatant fromthe settled precipitation mix.10. Thoroughly disperse the particles in the remaining solution.11. Immediately place 4 μl of the dispersed tungsten-DNA preparation inthe center of the support screen in a 13 mm plastic syringe filterholder.12. Attach the filter holder to the helium outlet in the target chamber.13. Replace the lid over the target tissue with a sterile protectivescreen. Place the sample into the target chamber, centered 16.5 cm underthe particle source and close the door.14. Open the valve to the vacuum source. When chamber vacuum reaches 28″of mercury, press the button to apply the accelerating gas pulse, whichdischarges the particles into the target chamber.15. Close the valve to the vacuum source. Allow air to return slowlyinto the target chamber through a sterilizing filter. Open the door,cover the sample with a sterile lid and remove the sample dish from thechamber.16. Repeat steps 10-15 for consecutive target plates using the sameprecipitation mix, filter and screen.17. Approximately 4 hours after bombardment, transfer the callus piecesfrom MSC3Osm to MSC3.18. Two days after shooting, transfer the callus onto selection medium.During this transfer, divide the callus into pieces ˜5 mm in diameter,with each piece being kept separate throughout the selection process.19. Subculture callus pieces at 2-3 week intervals.20. When callus pieces grow to ˜5 to 10 mm in diameter (typically 8 to12 weeks after bombardment) transfer onto regeneration medium at 28° C.in the light.21. When regenerated shoots are 30-60 mm high with severalwell-developed roots, transfer them into potting mix with the usualprecautions against mechanical damage, pathogen attack and desiccationuntil plantlets are established in the greenhouse.

Example 10 Soybean Embryo Transformation

Soybean embryos are bombarded with a plasmid containing a dimerizationdomain sequence operably linked to an ubiquitin promoter as follows. Toinduce somatic embryos, cotyledons, 3-5 mm in length dissected fromsurface-sterilized, immature seeds of the soybean cultivar A2872, arecultured in the light or dark at 26° C. on an appropriate agar mediumfor six to ten weeks. Somatic embryos producing secondary embryos arethen excised and placed into a suitable liquid medium. After repeatedselection for clusters of somatic embryos that multiplied as early,globular-staged embryos, the suspensions are maintained as describedbelow.

Soybean embryogenic suspension cultures can be maintained in 35 mlliquid media on a rotary shaker, 150 rpm, at 26° C. with florescentlights on a 16:8 hour day/night schedule. Cultures are subcultured everytwo weeks by inoculating approximately 35 mg of tissue into 35 ml ofliquid medium.

Soybean embryogenic suspension cultures may then be transformed by themethod of particle gun bombardment (Klein, et al., (1987) Nature(London) 327:70-73, U.S. Pat. No. 4,945,050). A Du Pont BiolisticPDS1000/HE instrument (helium retrofit) can be used for thesetransformations.

A selectable marker gene that can be used to facilitate soybeantransformation is a transgene composed of the 35S promoter fromCauliflower Mosaic Virus (Odell, et al., (1985) Nature 313:810-812), thehygromycin phosphotransferase gene from plasmid pJR225 (from E. coli;Gritz, et al., (1983) Gene 25:179-188) and the 3′ region of the nopalinesynthase gene from the T-DNA of the Ti plasmid of Agrobacteriumtumefaciens. The expression cassette comprising a dimerization domainsense sequence operably linked to the ubiquitin promoter can be isolatedas a restriction fragment. This fragment can then be inserted into aunique restriction site of the vector carrying the marker gene.

To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (inorder): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl₂(2.5 M). The particle preparation is then agitated for three minutes,spun in a microfuge for 10 seconds and the supernatant removed. TheDNA-coated particles are then washed once in 400 μl 70% ethanol andresuspended in 40 μl of anhydrous ethanol. The DNA/particle suspensioncan be sonicated three times for one second each. Five microliters ofthe DNA-coated gold particles are then loaded on each macro carrierdisk.

Approximately 300-400 mg of a two-week-old suspension culture is placedin an empty 60×15 mm petri dish and the residual liquid removed from thetissue with a pipette. For each transformation experiment, approximately5-10 plates of tissue are normally bombarded. Membrane rupture pressureis set at 1100 psi, and the chamber is evacuated to a vacuum of 28inches mercury. The tissue is placed approximately 3.5 inches away fromthe retaining screen and bombarded three times. Following bombardment,the tissue can be divided in half and placed back into liquid andcultured as described above.

Five to seven days post bombardment, the liquid media may be exchangedwith fresh media and eleven to twelve days post-bombardment with freshmedia containing 50 mg/ml hygromycin. This selective media can berefreshed weekly. Seven to eight weeks post-bombardment, green,transformed tissue may be observed growing from untransformed, necroticembryogenic clusters. Isolated green tissue is removed and inoculatedinto individual flasks to generate new, clonally propagated, transformedembryogenic suspension cultures. Each new line may be treated as anindependent transformation event. These suspensions can then besubcultured and maintained as clusters of immature embryos orregenerated into whole plants by maturation and germination ofindividual somatic embryos.

Example 11 Sunflower Meristem Tissue Transformation

Sunflower meristem tissues are transformed with an expression cassettecontaining a dimerization domain sequence operably linked to a ubiquitinpromoter as follows (see also, EP Patent Number 0 486233, hereinincorporated by reference and Malone-Schoneberg, et al., (1994) PlantScience 103:199-207). Mature sunflower seed (Helianthus annuus L.) aredehulled using a single wheat-head thresher. Seeds are surfacesterilized for 30 minutes in a 20% Clorox® bleach solution with theaddition of two drops of Tween® 20 per 50 ml of solution. The seeds arerinsed twice with sterile distilled water.

Split embryonic axis explants are prepared by a modification ofprocedures described by Schrammeijer, et al., (Schrammeijer, et al.,(1990) Plant Cell Rep. 9:55-60). Seeds are imbibed in distilled waterfor 60 minutes following the surface sterilization procedure. Thecotyledons of each seed are then broken off, producing a clean fractureat the plane of the embryonic axis. Following excision of the root tip,the explants are bisected longitudinally between the primordial leaves.The two halves are placed, cut surface up, on GBA medium consisting ofMurashige and Skoog mineral elements (Murashige, et al., (1962) Physiol.Plant., 15:473-497), Shepard's vitamin additions (Shepard (1980) inEmergent Techniques for the Genetic Improvement of Crops (University ofMinnesota Press, St. Paul, Minn.), 40 mg/l adenine sulfate, 30 g/lsucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25 mg/l indole-3-aceticacid (IAA), 0.1 mg/l gibberellic acid (GA₃), pH 5.6 and 8 g/l Phytagar.

The explants are subjected to microprojectile bombardment prior toAgrobacterium treatment (Bidney, et al., (1992) Plant Mol. Biol.18:301-313). Thirty to forty explants are placed in a circle at thecenter of a 60×20 mm plate for this treatment. Approximately 4.7 mg of1.8 mm tungsten microprojectiles are resuspended in 25 ml of sterile TEbuffer (10 mM Tris HCl, 1 mM EDTA, pH 8.0) and 1.5 ml aliquots are usedper bombardment. Each plate is bombarded twice through a 150 mm nytexscreen placed 2 cm above the samples in a PDS 1000® particleacceleration device.

Disarmed Agrobacterium tumefaciens strain EHA105 is used in alltransformation experiments. A binary plasmid vector comprising theexpression cassette that contains the dimerization domain gene operablylinked to the ubiquitin promoter is introduced into Agrobacterium strainEHA105 via freeze-thawing as described by Holsters, et al., (1978) Mol.Gen. Genet. 163:181-187. This plasmid further comprises a kanamycinselectable marker gene (i.e, nptII). Bacteria for plant transformationexperiments are grown overnight (28° C. and 100 RPM continuousagitation) in liquid YEP medium (10 gm/l yeast extract, 10 gm/l Bacto®peptone, and 5 gm/l NaCl, pH 7.0) with the appropriate antibioticsrequired for bacterial strain and binary plasmid maintenance. Thesuspension is used when it reaches an OD₆₀₀ of about 0.4 to 0.8. TheAgrobacterium cells are pelleted and resuspended at a final OD₆₀₀ of 0.5in an inoculation medium comprised of 12.5 mM MES pH 5.7, 1 gm/l NH₄Cl,and 0.3 gm/l MgSO₄.

Freshly bombarded explants are placed in an Agrobacterium suspension,mixed, and left undisturbed for 30 minutes. The explants are thentransferred to GBA medium and co-cultivated, cut surface down, at 26° C.and 18-hour days. After three days of co-cultivation, the explants aretransferred to 374B (GBA medium lacking growth regulators and a reducedsucrose level of 1%) supplemented with 250 mg/l cefotaxime and 50 mg/lkanamycin sulfate. The explants are cultured for two to five weeks onselection and then transferred to fresh 374B medium lacking kanamycinfor one to two weeks of continued development. Explants withdifferentiating, antibiotic-resistant areas of growth that have notproduced shoots suitable for excision are transferred to GBA mediumcontaining 250 mg/l cefotaxime for a second 3-day phytohormonetreatment. Leaf samples from green, kanamycin-resistant shoots areassayed for the presence of NPTII by ELISA and for the presence oftransgene expression by assaying for a modulation in meristemdevelopment (i.e., an alteration of size and appearance of shoot andfloral meristems).

NPTII-positive shoots are grafted to Pioneer® hybrid 6440 in vitro-grownsunflower seedling rootstock. Surface sterilized seeds are germinated in48-0 medium (half-strength Murashige and Skoog salts, 0.5% sucrose, 0.3%Gelrite®, pH 5.6) and grown under conditions described for explantculture. The upper portion of the seedling is removed, a 1 cm verticalslice is made in the hypocotyl and the transformed shoot inserted intothe cut. The entire area is wrapped with Parafilm® to secure the shoot.Grafted plants can be transferred to soil following one week of in vitroculture. Grafts in soil are maintained under high humidity conditionsfollowed by a slow acclimatization to the greenhouse environment.Transformed sectors of T₀ plants (parental generation) maturing in thegreenhouse are identified by NPTII ELISA and/or by dimerization domainactivity analysis of leaf extracts while transgenic seeds harvested fromNPTII-positive T₀ plants are identified by dimerization domain activityanalysis of small portions of dry seed cotyledon.

An alternative sunflower transformation protocol allows the recovery oftransgenic progeny without the use of chemical selection pressure. Seedsare dehulled and surface-sterilized for 20 minutes in a 20% Clorox®bleach solution with the addition of two to three drops of Tween® 20 per100 ml of solution, then rinsed three times with distilled water.Sterilized seeds are imbibed in the dark at 26° C. for 20 hours onfilter paper moistened with water. The cotyledons and root radical areremoved and the meristem explants are cultured on 374E (GBA mediumconsisting of MS salts, Shepard vitamins, 40 mg/l adenine sulfate, 3%sucrose, 0.5 mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/l GA, and 0.8% Phytagarat pH 5.6) for 24 hours under the dark. The primary leaves are removedto expose the apical meristem, around 40 explants are placed with theapical dome facing upward in a 2 cm circle in the center of 374M (GBAmedium with 1.2% Phytagar) and then cultured on the medium for 24 hoursin the dark.

Approximately 18.8 mg of 1.8 μm tungsten particles are resuspended in150 μl absolute ethanol. After sonication, 8 μl of it is dropped on thecenter of the surface of macrocarrier. Each plate is bombarded twicewith 650 psi rupture discs in the first shelf at 26 mm of Hg helium gunvacuum.

The plasmid of interest is introduced into Agrobacterium tumefaciensstrain EHA105 via freeze thawing as described previously. The pellet ofovernight-grown bacteria at 28° C. in a liquid YEP medium (10 g/l yeastextract, 10 g/l Bacto® peptone and 5 g/l NaCl, pH 7.0) in the presenceof 50 μg/l kanamycin is resuspended in an inoculation medium (12.5 mM2-mM 2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH₄CI and 0.3 g/IMgSO₄ at pH 5.7) to reach a final concentration of 4.0 at OD₆₀₀.Particle-bombarded explants are transferred to GBA medium (374E) and adroplet of bacteria suspension is placed directly onto the top of themeristem. The explants are co-cultivated on the medium for 4 days, afterwhich the explants are transferred to 374C medium (GBA with 1% sucroseand no BAP, IAA, GA3 and supplemented with 250 μg/ml cefotaxime). Theplantlets are cultured on the medium for about two weeks under 16-hourday and 26° C. incubation conditions.

Explants (around 2 cm long) from two weeks of culture in 374C medium arescreened for a modulation in meristem development (i.e., an alterationof size and appearance of shoot and floral meristems). After positive(i.e., a change in dimerization domain expression) explants areidentified, those shoots that fail to exhibit an alteration indimerization domain activity are discarded and every positive explant issubdivided into nodal explants. One nodal explant contains at least onepotential node. The nodal segments are cultured on GBA medium for threeto four days to promote the formation of auxiliary buds from each node.Then they are transferred to 374C medium and allowed to develop for anadditional four weeks. Developing buds are separated and cultured for anadditional four weeks on 374C medium. Pooled leaf samples from eachnewly recovered shoot are screened again by the appropriate proteinactivity assay. At this time, the positive shoots recovered from asingle node will generally have been enriched in the transgenic sectordetected in the initial assay prior to nodal culture.

Recovered shoots positive for altered dimerization domain expression aregrafted to Pioneer hybrid 6440 in vitro-grown sunflower seedlingrootstock. The rootstocks are prepared in the following manner. Seedsare dehulled and surface-sterilized for 20 minutes in a 20% Clorox®bleach solution with the addition of two to three drops of Tween® 20 per100 ml of solution, and are rinsed three times with distilled water. Thesterilized seeds are germinated on the filter moistened with water forthree days, then they are transferred into 48 medium (half-strength MSsalt, 0.5% sucrose, 0.3% Gelrite® pH 5.0) and grown at 26° C. under thedark for three days, then incubated at 16-hour-day culture conditions.The upper portion of selected seedling is removed, a vertical slice ismade in each hypocotyl and a transformed shoot is inserted into 8V-Cut.The cut area is wrapped with Parafilm®. After one week of culture on themedium, grafted plants are transferred to soil. In the first two weeks,they are maintained under high humidity conditions to acclimatize to agreenhouse environment.

Example 12 Agrobacterium Mediated Grass Transformation

Grass plants may be transformed by following the Agrobacterium mediatedtransformation of Luo, et al., (2004) Plant Cell Rep (2004) 22:645-652.

Materials and Methods Plant Material

A commercial cultivar of creeping bentgrass (Agrostis stolonifera L.,cv. Penn-A-4) supplied by Turf-Seed (Hubbard, Ore.) can be used. Seedsare stored at 4° C. until used.

Bacterial Strains and Plasmids

Agrobacterium strains containing one of 3 vectors are used. One vectorincludes a pUbi-gus/Act1-hyg construct consisting of the maize ubiquitin(ubi) promoter driving an intron-containing b-glucuronidase (GUS)reporter gene and the rice actin 1 promoter driving a hygromycin (hyg)resistance gene. The other two pTAP-arts/35S-bar andpTAP-barnase/Ubi-bar constructs are vectors containing a ricetapetum-specific promoter driving either a rice tapetum-specificantisense gene, rts (Lee, et al., (1996) Int Rice Res Newsl 21:2-3) or aribonuclease gene, barnase (Hartley, (1988) J Mol Biol 202:913-915),linked to the cauliflower mosaic virus 35S promoter (CaMV 35S) or therice ubi promoter (Huq, et al, (1997) Plant Physiol 113:305) driving thebar gene for herbicide resistance as the selectable marker.

Induction of Embryogenic Callus and Agrobacterium-MediatedTransformation

Mature seeds are dehusked with sand paper and surface sterilized in 10%(v/v) Clorox® bleach (6% sodium hypochlorite) plus 0.2% (v/v) Tween® 20(Polysorbate 20) with vigorous shaking for 90 min. Following rinsingfive times in sterile distilled water, the seeds are placed ontocallus-induction medium containing MS basal salts and vitamins(Murashige and Skoog, (1962) Physiol Plant 15:473-497), 30 g/l sucrose,500 mg/l casein hydrolysate, 6.6 mg/l 3,6-dichloro-o-anisic acid(dicamba), 0.5 mg/l 6-benzylaminopurine (BAP) and 2 g/l Phytagel. The pHof the medium is adjusted to 5.7 before autoclaving at 120° C. for 20min. The culture plates containing prepared seed explants are kept inthe dark at room temperature for 6 weeks. Embryogenic calli are visuallyselected and subcultured on fresh callus-induction medium in the dark atroom temperature for 1 week before co-cultivation.

Transformation

The transformation process is divided into five sequential steps:agro-infection, co-cultivation, antibiotic treatment, selection andplant regeneration. One day prior to agro-infection, the embryogeniccallus is divided into 1- to 2-mm pieces and placed on callus-inductionmedium containing 100 μM acetosyringone. A 10-ml aliquot ofAgrobacterium suspension (OD=1.0 at 660 nm) is then applied to eachpiece of callus, followed by 3 days of co-cultivation in the dark at 25°C. For the antibiotic treatment step, the callus is then transferred andcultured for 2 weeks on callus-induction medium plus 125 mg/l cefotaximeand 250 mg/l carbenicillin to suppress bacterial growth. Subsequently,for selection, the callus is moved to callus-induction medium containing250 mg/l cefotaxime and 10 mg/l phosphinothricin (PPT) or 200 mg/lhygromycin for 8 weeks. Antibiotic treatment and the entire selectionprocess is performed at room temperature in the dark. The subcultureinterval during selection is typically 3 weeks. For plant regeneration,the PPT- or hygromycin-resistant proliferating callus is first moved toregeneration medium (MS basal medium, 30 g/l sucrose, 100 mg/lmyo-inositol, 1 mg/l BAP and 2 WI Phytagel) supplemented withcefotaxime. PPT or hygromycin. These calli are kept in the dark at roomtemperature for 1 week and then moved into the light for 2-3 weeks todevelop shoots. Small shoots are then separated and transferred tohormone-free regeneration medium containing PPT or hygromycin andcefotaxime to promote root growth while maintaining selection pressureand suppressing any remaining Agrobacterium cells. Plantlets withwell-developed roots (3-5 weeks) are then transferred to soil and growneither in the greenhouse or in the field.

Staining for GUS Activity

GUS activity in transformed callus is assayed by histochemical stainingwith 1 mM 5-bromo-4-chloro-3-indolyl-b-d-glucuronic acid (X-Gluc,Biosynth, Staad, Switzerland) as described in Jefferson, (1987) PlantMol Biol Rep 5:387-405. The hygromycin-resistant callus surviving fromselection was incubated at 37 C overnight in 100 μl of reaction buffercontaining X-Gluc. GUS expression is then documented by photography.

Vernalization and Out-Crossing of Transgenic Plants

Transgenic plants are maintained out of doors in a containment nursery(3-6 months) until the winter solstice in December. The vernalizedplants are then transferred to the greenhouse and kept at 25° C. under a16/8 h [day/light (artificial light)] photoperiod and surrounded bynon-transgenic wild-type plants that physically isolated them from otherpollen sources. The plants will initiate flowering 3-4 weeks after beingmoved back into the greenhouse. They are out-crossed with the pollenfrom the surrounding wild-type plants. The seeds collected from eachindividual transgenic plant are germinated in soil at 25° C. and Tiplants are grown in the greenhouse for further analysis.

Seed Testing

Test of the Transgenic Plants and their Progeny for Resistance to PPT

Transgenic plants and their progeny are evaluated for tolerance toglufosinate (PPT) indicating functional expression of the bar gene. Theseedlings are sprayed twice at concentrations of 1-10% (v/v) Finale©(AgrEvo USA, Montvale, N.J.) containing 11% glufosinate as the activeingredient. Resistant and sensitive seedlings are clearlydistinguishable 1 week after the application of Finale© in all thesprayings.

Statistical Analysis

Transformation efficiency for a given experiment is estimated by thenumber of PPT-resistant events recovered per 100 embryogenic calliinfected and regeneration efficiency is determined using the number ofregenerated events per 100 events attempted. The mean transformation andregeneration efficiencies are determined based on the data obtained frommultiple independent experiments. A Chi-square test can be used todetermine whether the segregation ratios observed among Ti progeny forthe inheritance of the bar gene as a single locus fit the expected 1:1ratio when out-crossed with pollen from untransformed wild-type plants.

DNA Extraction and Analysis

Genomic DNA is extracted from approximately 0.5-2 g of fresh leavesessentially as described by Luo, et al., (1995) Mol Breed 1:51-63. Tenmicrograms of DNA is digested with HindIII or BamHI according to thesupplier's instructions (New England Biolabs, Beverly, Mass.). Fragmentsare size-separated through a 1.0% (w/v) agarose gel and blotted onto aHybond-N+ membrane (Amersham Biosciences, Piscataway, N.J.). The bargene, isolated by restriction digestion from pTAP-arts/35S-bar, is usedas a probe for Southern blot analysis. The DNA fragment is radiolabeledusing a Random Priming Labeling kit (Amersham Biosciences) and theSouthern blots are processed as described by Sambrook, et al., (1989)Molecular cloning: a laboratory manual, 2nd edn, Cold Spring HarborLaboratory Press, New York.

Polymerase Chain Reaction

The two primers designed to amplify the bar gene are as follows:5-GTCTGCACCATCGTCAACC-3′ (SEQ ID NO: 42), corresponding to the proximityof the 5′ end of the bar gene and 5′-GAAGTCCAGCTGCCAGAAACC-3′ (SEQ IDNO: 43), corresponding to the 3′ end of the bar coding region. Theamplification of the bar gene using this pair of primers should resultin a product of 0.44 kb. The reaction mixtures (25 μl total volume)consist of 50 mM KCl, 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, 0.1% (w/v)Triton X-100, 200 μM each of dATP, dCTP, dGTP and dTTP, 0.5 μM of eachprimer, 0.2 μg of template DNA and 1 U Taq DNA polymerase (QIAGEN,Valencia, Calif.). Amplification is performed in a Stratagene RobocyclerGradient 96 thermal cycler (La Jolla, Calif.) programmed for 25 cyclesof 1 min at 94° C. (denaturation), 2 min at 55° C. (hybridization), 3min at 72° C. (elongation) and a final elongation step at 72° C. for 10min. PCR products are separated on a 1.5% (w/v) agarose gel and detectedby staining with ethidium bromide.

Example 13 Plant Characterization Analysis—Greenhouse

Greenhouse experiments were performed with two constructs plus acomparative control. All had 35s::BAR as the selectable marker. Php37407contained S2A PRO::D8 MPL+F3.7 PRO::CESA4+FTM1 PRO::DD+NAS2 PRO::DD.Php39175 contained S2A PRO::D8 MPL S89T (ALT4). For each construct, 10events were planted. An equal number of positive and negative plants (4per week×4 weeks) were expected. Due to greenhouse growth conditions andsubsequent extra plantings, the outcome of samples was:

For php37407: 28 positive plants from 10 events and 24 negative siblingsfrom 9 events.For php39175: 25 positive plants from 8 events and 29 negative siblingsfrom 10 events.

Observations were performed on each of the plants, and measurementsrecorded. Data collected included: Plant Height, Leaf Width and LeafLength (leaves −2, +2, +4 from ear node), Central Tassel SpikeMeasurement (absolute value and normalized to height), Anther ExertionLength, Tassel Score (1-9: 1 being very small with no branches, 5average size with approximately 6 branches, 9 very large with 20 or morebranches), Pollen Score (1-5: low to high, a measurement of collectedpollen, each unit equivalent to 0.7″ of collected pollen in a 0.25″ wideapparatus) and Leaf Count.

Final analysis of the plants showed that the stacked php37407 constructcontaining the dimerization domain had moderating effects on the dwarfgene's phenotype exhibited in php39175 plants. Plant height for php37407increased 8.8% as compared with php39175. The increases in the leafwidth and the reductions in leaf length with the dwarf gene in php39175were also moderated with the stack in php37407. In the php37407 plants,leaf width was reduced 6% in the leaf two nodes below the ear, 4.2% inthe leaf two nodes above the ear and 5.4% in the leaf four nodes abovethe ear. The leaf lengths in the php37407 plants were increased 2.7% and2.8% respectively for the nodes two below and two above the ear andshowed no difference for the leaf four nodes above the ear.Additionally, the absolute length of the central tassel spike wasincreased by 3% in the php37407 samples as compared with the php39175samples. The tassel length as a percentage of height was reduced by 6.4%in php37407, moderating the dwarf gene's effect to increase the relativetassel length compared to the vegetative plant height. Furthermore, on arepresentative subset of samples, the exerted anther length includingthe filament plus anther in php37407 plants was increased by 9.9% ascompared with the php39175 anthers. In addition, the tassel score index(1-9) taken by the greenhouse in the stacked php37407 plants showed anincrease of 9.5%. The pollen score index (1-5) on a representativesubset of samples showed no difference between the samples. Leaf countswere also similar between php37407 and php39175 and found one nodegreater per plant as compared with their negative siblings. Overall, thestack of genes in the php37407 plants displayed a moderating effect ofthe dwarf gene phenotype in the php39175 samples. Moderating the effectof the dwarf gene included, but was not limited to: increased plantheight and tassel size, leaf length and anther exerted.

Example 14 Arabidopsis Dimerization Domain Study

Arabidopsis plants ecotype Columbia were transformed with a constructcontaining a constitutive promoter or tissue-preferred promoter drivingexpression of the dimerization domain (DD). Plants were transformedusing Agrobacterium-mediated transformation method and positivetransformants were selected by resistance to an herbicide. TransgenicArabidopsis plants were grown in nutrient-rich soil under greenhouseconditions. Seeds were collected to determine improvements in yield oryield-related traits between transgenic plants and control plants.Control plants are positive transformants that contain the vectorbackbone without the promoter and dimerization domain. Effectivetransgenic events are those that show an increase in yield or seedweight under normal growing conditions.

Transgenic plants containing a putative leaf-preferred promoter drivingexpression of the dimerization domain showed an approximately 22%increase in seed weight over control plants.

Example 15 Root Growth Analysis

Seed segregating for transgene heterozygote and wild type are planted inCustom 200C pot filled with Turface MVP then watered with nutrientsolution containing 1 mM KNO3 or 4 mM KNO3 as nitrogen source along witha full complement of other nutrients:

Nutrient 1 mM KNO₃ 4 mM KNO₃ 10x Micron utrients 400 ml 400 ml KH₂PO₄136.02 Mwt 272 g 272 g MgSO₄ 120.36 Mwt 963 g 963 g KNO₃ fertilizergrade 400 g 1200 g KCl 74.55 Mwt 596 g — *CaCl₂ 147.01 Mwt 588 g 588 gSprint 330 335 g 335 g / 100 l / 100 lAdd 84 ml H₂SO₄ to reduce pH. Optimum pH is 5-5.5. Add 200 ul of thenutrient solution to 3 ml tap water and check the pH, it should be5-5.8. If distilled water is used the pH will have to be raised with 10MKOH instead of decreased.*If using tap water with Ca⁺⁺ concentration in the 0.5-0.7 mM levelreduce this amount to 235 g. If comparing 6 mM growth to any othernutrient mix maintain the CaCl₂ level at 588 g/100 l.

10x Micronutrients Stock solution mg/liter  15 mM H₃BO₃ 1852 mg   5 mMMnCl₂•4H₂O 1980 mg   5 mM ZnSO₄•7 H₂O 2874 mg 0.5 mM CuSO₄•5H₂O  250 mg0.5 mM H₂MoO₄•H₂O  242 mg

After 3 weeks of growth in these media SPAD meter measurements are madeby averaging at least 5 readings taken from the base of the youngestmost fully expanded leaf. Plants are removed from the pots, the Turfacewashed from the roots and separated into shoots and roots. These samplesare dried (70° C. for 72 hr) and dried roots are weighed separately fromthe shoots. The dried shoots are ground to a fine powder and total Ndetermined using a sample of the ground tissue. From these parametersgreenness (SPAD), total plant weight, shoot weight, root weight,root/shoot ratio, shoot nitrogen concentration and total N arecalculated for low and high N fertility grown plants.

Plants have a higher root/shoot ratio when grown in lower nitrogenfertility. Agronomic conditions for growing maize have higher soilnitrate conditions when the plants are the smallest. Higher soil nitrateconditions favor lower root/shoot ratios which does not favor extensivesoil exploration by roots. These transgenes that increase the root/shootratio under high or low nitrogen fertility would likely explore agreater portion of the soil early during growth and maximize plantgrowth. Root/shoot ratios would be higher in higher N fertilities.

The use of a root preferred promoter such as NAS2 and the dimerizationdomain (DD) enhances root growth early in development. The changes inroot growth can be detected at the tissue culture stages of plantregeneration following transformation with this gene specifically by theappearance of more roots and larger diameter roots in test tubesprepared for rooting (ref. Zhao). Transgenic seed expressing NAS2:DDwould be expected to have an enhanced early root growth phenotypesimilar to that observed in tissue culture experiments. The expectedphenotype in the assay mentioned above would be expected to produce ahigher root dry weight at the end of the growth period of three weeks.An altered root growth (higher) would be especially desirable underhigher N conditions because of a greater soil exploration capacity intransgenic versus non transgenic plants.

Example 16 The Use of DD (Dimerization Domain) Components with ModerateDwarfing Genes (D8MPL) to Improve Creeping Bentgrass (Agrostisstolonifera L.) for Turf Grass Applications

The semi-dwarf characteristics of S2a:D8MPL in corn could be used toimprove turf grass species such as creeping bentgrass (Agrostisstolonifera L). Specifically, a more compact leaf with increased widthand reduced length is desirable and the dark green leaf color observedin corn would be especially desirable in turf grass. In addition to thereduced leaf length with S2a:D8MPL, roots may also have shorter lengthcompared to non transgenic creeping bent grass. The use of the DDdominant negative transgene with a root preferred promoter such as NAS2could be combined in a transgene stack to selectively increase the rootgrowth relative to a more compact leaf phenotype desired in the leaves.The compact leaf structure would also have advantages in terms ofreduced maintenance (mowing) with similar or reduced amounts of addedfertilizer. Furthermore, the use of the DD with a root preferred orspecific promoter would increase the relative root length and rootdensity compared to the expectation of smaller roots with a dwarfshoot/leaf phenotype. Increasing root length and density, especiallyearlier in plant development, would aide establishment and could alsomoderate irrigation requirements for establishment and maintenance ofcommercial turf grass plantings. Similar advantages are anticipated fornon-commercial home use of transgenic Agrostis species—ease inestablishment because of strong root formation from seedlings and moreefficient maintenance in terms of less mowing and irrigation to maintaina desirable turfgrass (i.e., dark green) appearance above ground anddeeper more vigorous roots to support leaf growth and turf qualitymaintenance.

Example 17 Variants of Dimerization Domain Sequences

A. Variant Nucleotide Sequences of Dimerization Domain that do not Alterthe Encoded Amino Acid Sequence

The dimerization domain nucleotide sequences are used to generatevariant nucleotide sequences having the nucleotide sequence of the openreading frame with about 70%, 75%, 80%, 85%, 90% and 95% nucleotidesequence identity when compared to the starting unaltered ORF nucleotidesequence of the corresponding SEQ ID NO. These functional variants aregenerated using a standard codon table. While the nucleotide sequence ofthe variants are altered, the amino acid sequence encoded by the openreading frames do not change. These variants are associated with theability of the dimerization domain to form defective dimmers therebypreventing the inhibitory response to GA.

B. Variant Amino Acid Sequences of Dimerization Domain Polypeptides

Variant amino acid sequences of the dimerization domain polypeptides aregenerated. In this example, one amino acid is altered. Specifically, theopen reading frames are reviewed to determine the appropriate amino acidalteration. The selection of the amino acid to change is made byconsulting the protein alignment (with the other orthologs and othergene family members from various species). An amino acid is selectedthat is deemed not to be under high selection pressure (not highlyconserved) and which is rather easily substituted by an amino acid withsimilar chemical characteristics (i.e., similar functional side-chain).Using a protein alignment, an appropriate amino acid can be changed.Once the targeted amino acid is identified, the procedure outlined inthe following section C is followed. Variants having about 70%, 75%,80%, 85%, 90% and 95% nucleic acid sequence identity are generated usingthis method.

C. Additional Variant Amino Acid Sequences of Dimerization DomainPolypeptides

In this example, artificial protein sequences are created having 80%,85%, 90% and 95% identity relative to the reference protein sequence.This latter effort requires identifying conserved and variable regionsfrom an alignment and then the judicious application of an amino acidsubstitutions table. These parts will be discussed in more detail below.

Largely, the determination of which amino acid sequences are altered ismade based on the conserved regions among dimerization domain protein oramong the other dimerization domain polypeptides. It is recognized thatconservative substitutions can be made in the conserved regions belowwithout altering function. In addition, one of skill will understandthat functional variants of the dimerization domain sequence of theinvention can have minor non-conserved amino acid alterations in theconserved domain.

Artificial protein sequences are then created that are different fromthe original in the intervals of 80-85%, 85-90%, 90-95% and 95-100%identity. Midpoints of these intervals are targeted, with liberallatitude of plus or minus 1%, for example. The amino acids substitutionswill be effected by a custom Perl script. The substitution table isprovided below in Table 5.

TABLE 5 Substitution Table Strongly Similar and Rank of Optimal Order toAmino Acid Substitution Change Comment I L, V 1 50:50 substitution L I,V 2 50:50 substitution V I, L 3 50:50 substitution A G 4 G A 5 D E 6 E D7 W Y 8 Y W 9 S T 10 T S 11 K R 12 R K 13 N Q 14 Q N 15 F Y 16 M L 17First methionine cannot change H Na No good substitutes C Na No goodsubstitutes P Na No good substitutes

First, any conserved amino acids in the protein that should not bechanged is identified and “marked off” for insulation from thesubstitution. The start methionine will of course be added to this listautomatically. Next, the changes are made.

H, C and P are not changed in any circumstance. The changes will occurwith isoleucine first, sweeping N-terminal to C-terminal. Then leucine,and so on down the list until the desired target it reached. Interimnumber substitutions can be made so as not to cause reversal of changes.The list is ordered 1-17, so start with as many isoleucine changes asneeded before leucine, and so on down to methionine. Clearly many aminoacids will in this manner not need to be changed. L, I and V willinvolve a 50:50 substitution of the two alternate optimal substitutions.

The variant amino acid sequences are written as output. Perl script isused to calculate the percent identities. Using this procedure, variantsof the dimerization domain polypeptides are generating having about 80%,85%, 90% and 95% amino acid identity to the starting unaltered ORFnucleotide sequence of SEQ ID NO: 9.

All publications and patent applications are herein incorporated byreference to the same extent as if each individual publication or patentapplication was specifically and individually indicated by reference.

The invention has been described with reference to various specific andpreferred embodiments and techniques. However, it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention.

What is claimed is:
 1. An isolated nucleic acid encoding a dimerizationdomain, the dimerization domain comprising a consensus amino acidsequence of SEQ ID NO: 41 or a sequence that is 90% identical to SEQ IDNO:
 41. 2. The nucleic acid of claim 1 encoding a dimerization domain,the dimerization domain consisting essentially of the amino acidsequence of SEQ ID NO: 19 or 21 or a sequence that is 90% identical toSEQ ID NO: 19 or
 21. 3. The isolated nucleic acid of claim 1 comprisinga polynucleotide sequence of SEQ ID NO:
 9. 4. The isolated nucleic acidof claim 1, wherein the dimerization domain binds to a native maize D8protein or D9 protein to produce a nonfunctional D8 or D9 dimer.
 5. Arecombinant expression cassette, comprising the polynucleotide of claim3, wherein the polynucleotide is operably linked to a promoter.
 6. Ahost cell comprising the expression cassette of claim
 5. 7. A transgenicplant comprising the recombinant expression cassette of claim
 5. 8. Thetransgenic plant of claim 7, wherein said plant is a monocot.
 9. Thetransgenic plant of claim 7, wherein said plant is a dicot.
 10. Thetransgenic plant of claim 7, wherein said plant is selected from thegroup consisting of: maize, soybean, sunflower, sorghum, canola, wheat,alfalfa, cotton, rice, barley, millet, peanut, sugar cane, grass,turfgrass and cocoa.
 11. A transgenic seed from the transgenic plant ofclaim
 7. 12. A method of modulating harvest index in a transgenic plant,the method comprising expressing a recombinant polynucleotide encoding adimerization domain of a dwarf gene.
 13. The method of claim 12, whereinthe dwarf gene is D8 from maize.
 14. The method of claim 12, wherein thepolynucleotide comprises the nucleic acid sequence of claim 3 operablylinked to a promoter.
 15. The method of claim 12, wherein the plant isselected from the group consisting of: maize, soybean, sunflower,sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet, peanut,sugar cane, grass, turfgrass and cocoa.
 16. The method of claim 12,wherein the dimerization domain forms a non-functional dimer ofendogenous maize D8 or D9 protein.
 17. A method of modulating planttissue growth with a dimerization domain in a plant, comprisingexpressing a recombinant expression cassette comprising thepolynucleotide of claim 3 operably linked to a promoter.
 18. The methodof claim 17, wherein plant tissue growth is due to reduced inhibition byendogenous gibberellic acid.
 19. The method of claim 17, wherein theplant is selected from the group consisting of: maize, soybean, sorghum,canola, wheat, alfalfa, cotton, rice, barley, millet, peanut, sugarcane, grass, turfgrass and cocoa.
 20. A product derived from the methodof processing of transgenic plant component expressing an isolatedpolynucleotide encoding a dimerization domain, the method comprising: a.growing a plant that expresses a polynucleotide having at least 90%sequence identity to the full length sequence of SEQ ID NO: 9, operablylinked to a promoter; and b. processing the plant component to obtain aproduct.
 21. The product of claim 20, wherein the plant component is aseed.
 22. A product according to claim 20, wherein the polynucleotidefurther encodes a polypeptide selected of SEQ ID NO:
 19. 23. A productaccording to claim 20, which is a constituent of ethanol.
 24. The methodof claim 12, wherein the plant has improved canopy shape.
 25. The methodof claim 12, wherein the plant has increased photosynthetic capacity inleaf tissue.
 26. The method of claim 12, wherein the plant has improvedstalk strength.
 27. The method of claim 12, wherein the plant hasimproved plant standibility.
 28. The method of claim 12, wherein theplant has altered vascular bundle structure or number.
 29. The method ofclaim 12, wherein the plant has increased root biomass.
 30. The methodof claim 12, wherein the plant has enhanced root growth.
 31. The methodof claim 12, wherein the plant has modulated shoot development.
 32. Themethod of claim 12, wherein the plant has modulated leaf development.33. The method of claim 12, wherein the plant has improved silagequality and digestibility.
 34. The method of claim 14, wherein thepromoter is selected from the group consisting of a leaf specificpromoter, vascular element preferred promoter and a root specificpromoter.